Sodium dodecyl sulfate compositions for inactivating prions

ABSTRACT

An antiseptic composition useful in destroying the infectivity of infectious proteins such as prions is disclosed. The antiseptic composition is preferably maintained at either a low pH of 4.0 or less or a high pH of 10.0 or more either of which allows for an environment under which the active component (which is preferably sodium dodecyl sulfate) destroys infectivity. The composition may be added to blood, blood products, collagen, tissues and organs prior to transplantation. The composition also may be added to livestock feed to denature any prions in the livestock. Methods of denaturing infectious proteins are also disclosed which method can use but do not require higher temperatures and long period of exposure.

CROSS-REFERENCES

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 09/904,178, filed Jul. 11, 2001 which is a continuation-in-partof U.S. application Ser. No. 09/699,284, filed Oct. 26, 2000, which is acontinuation-in-part of U.S. application Ser. No. 09/494,814, filed Jan.31, 2000, which is a continuation-in-part of U.S. application Ser. No.09/447,456, filed Nov. 22, 1999 now U.S. Pat. No. 6,331,296, which is acontinuation-in-part of U.S. application Ser. No. 09/322,903, filed Jun.1, 1999, now U.S. Pat. No. 6,214,366 and to which priority is claimedunder 35 U.S.C. § 120. This application is also a continuation-in-partof U.S. Pat. No. 6,221,614, issued Apr. 24, 2001, which is acontinuation-in-part of U.S. application Ser. No. 09/151,057, filed onSep. 10, 1998, which is a continuation-in-part of U.S. application Ser.No. 09/026,957, filed on Feb. 20, 1998, which is a continuation-in-partof U.S. application Ser. No. 08/804,536, filed on Feb. 21, 1997, nowU.S. Pat. No. 5,891,641, and to which priority is claimed under 35U.S.C. § 120.

GOVERNMENT SUPPORT

[0002] This work was supported, in part, by grants from the NationalInstitutes of Health NS14069, AG08967, AG02132, AG10770 and K08NS02048-02. The government may have certain rights in this work.

FIELD OF THE INVENTION

[0003] The present invention relates generally to compositions forinactivating infectious prions on infected surfaces and in a range ofdifferent products including, blood, organ and tissue products, foodproducts and livestock feed.

BACKGROUND OF THE INVENTION

[0004] Antiseptic compositions have been known for over 100 years. Inaddition to various compositions there are a range of different methods,which are known to be effective in killing bacteria and inactivatingviruses. Such methods include the use of high temperature, alone or incombination with radiation, over sufficient periods of time to killbacteria or disrupt viruses and thereby inactivate them. These methodsare extreme, and can damage sensitive medical equipment, thus decreasingits useful life.

[0005] Examples of fast acting topical antiseptic compositions aredisclosed n U.S. Pat. No. 6,110,908, issued Aug. 29, 2000. Anotherantibacterial composition is disclosed in U.S. Pat. No. 6,025,312,issued Feb. 15, 2000. Examples of other antiseptic compositions aretaught within U.S. Pat. No. 5,336,432, issued Aug. 9, 1994; U.S. Pat.No. 5,308,611, issued May 3, 1994; U.S. Pat. No. 6,106,773, issued Aug.22, 2000 and U.S. Pat. No. 6,096,216, issued Aug. 1, 2000.

[0006] Conventional antiseptic compositions and antiseptic methodologiesare generally insufficient for inactivating infectious proteins such asprions. Although prions can be inactivated by relatively hightemperatures over very long periods of time, the temperature ranges andtime periods generally used to kill bacteria and inactivate the virusesare insufficient to inactivate prions. One approach to solving thisproblem is to attempt to remove prions from solutions. A chromographicremoval process is disclosed within U.S. Pat. No. 5,808,011. Further,others have attempted to provide compositions and methodologies that areintended to inactivate prions as taught within U.S. Pat. No. 5,633,349.However, such processes generally take relatively long periods of time(e.g., more than 12 hours) and generally do not provide a solution thatcould be readily and economically utilized in order to inactivate prionson food products, biological materials, medical equipment, and livestockfeed.

[0007] The present invention offers antiseptic compositions and methods,which may be utilized under mild conditions, for inactivating prionsthat will not damage any existing equipment, food, or biologicalsubstance, as described below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008]FIG. 1 is a schematic drawing of a dendrimer molecule showing thedefined “generations” of homodisperse structure created using arepetitive divergent growth technique. The specific diagram is of PAMAM,generation 2.0 (ethylene diamine core).

[0009]FIG. 2A and FIG. 2B are photographs of gels, and FIG. 2C is agraph of data plotted to indicate survival rates. In FIG. 2A, lane 3shows results where no dendrimers were added and prions remain whereasdendrimers were added in lane 4 showing the removal of PrP^(Sc) presentin a cell culture. FIG. 2B shows that the use of dendrimers incompositions of the invention does not effect overall proteinexpression. FIG. 2C shows that when cells are assayed for prioninfectivity by injection into mice showing the use of dendrimers curescells.

[0010]FIG. 3 includes gel panels 3A and 3B and within FIG. 3A columns 3and 4 labeled +PK (proteinase K) show that dendrimers are effective inremoving prions best at a pH of less than 4. FIG. 3B shows that severaldifferent types of dendrimers are effective in inactivating prioninfectivity.

[0011]FIG. 4 includes double gel panel 4A and double gel panel 4B.Within the upper panel 4A there is a showing that different strains ofprions have different susceptibility to dendrimers indicating thatspecific dendrimers could be used to determine the type of infectivity(prion strain) in a sample with similar results shown in FIG. 4A.

[0012]FIG. 5 includes gel panels A, B, C and D. In FIG. 5 there is ashowing that the addition of urea enhances the ability of dendrimers todenature and remove prion infectivity. FIG. 5B indicates that theparticular dendrimers tested are most effective at approximately 37 C.FIGS. 5C and 5D show that dendrimer induced inactivation of prions isirreversible.

[0013]FIG. 6 includes high-resolution photographs 6A and 6B, whichprovide a visualization of what happens to prion rods exposed todendrimers.

[0014]FIG. 7 includes photographs of 7A, 7B and 7C using afluorescein-labeled PPI which demonstrates that dendrimers are effectiveinside lysosomes.

[0015]FIG. 8 includes gel panels 8A (2 hours) and 8B (5 minutes). WithinFIG. 8A column 4 (+) shows that SDS at a pH of about 3.3 completelyeliminates PrP^(C) whereas 1% SDS at a pH of 7.0 is not effective. FIG.8B within column 4 shows that 1% SDS at a pH of 3.3 is effective ininactivating prions after only five minutes of exposure.

[0016]FIG. 9 includes a gel panel wherein columns show results carriedout at different temperatures indicating that the optimal temperaturefor using acetic SDS to eliminate prions is greater than 20° C.

[0017]FIG. 10 shows that urea (column 4) is also effective ininactivating prions under acetic conditions.

[0018]FIG. 11 shows four separate gels with each of the gels run at ninedifferent specific pH levels showing that SDS does denature PrP^(Sc) atlow pH and high pH but not at a relatively neutral pH and furthershowing that increasing the percent concentration of SDS improves theability of the formulation to denature PrP^(Sc).

[0019]FIG. 12 provides gel panels that show that acetic buffers otherthan acetic acid and sodium acetate can be used in combination with SDSin order to denature PrP^(Sc).

[0020]FIG. 13A is a photograph of a gel that shows the denaturation ofPrP^(Sc) by different alkyl sulfates and alkyl sulfonates.

[0021]FIG. 13B is a photograph of a gel that shows the denaturation ofPrP^(Sc) by alkyl sulfates at 4° C. or 37° C.

[0022]FIG. 14 is a photograph of a Western blot gel. Homogenates ofBSE-infected bovine brain were successfully denatured of prions byexposure to acidic SDS, see lane 3.

SUMMARY OF THE INVENTION

[0023] Compositions or formulations are disclosed which when used in themethods of the invention inactivate the infectability of infectiousproteins such as prions under relatively mild conditions as compared tothe conditions generally thought to be needed to render infectiousproteins non-infectious. For example, conditions of 100° C. or more forhours are used to inactivate prions whereas the present invention allowsfor prion inactivation at mild temperatures such as 20° C. to 65° C. orless for 5 to 30 minutes or less.

[0024] A number of different active ingredients for the formulation aredisclosed including dendrimers and detergents. However, salts of alkylsulfates (e.g. sodium dodecyl sulfate) are preferred. The alkyl moietymay contain 2 to 40 carbon atoms but preferably contains 6 to 12 carbonatoms. Although the invention may be used to inactivate a range ofdifferent malformed or infectious proteins it is preferably used toinactivate the infectivity of infectious prions which may be in or onpharmaceutical formulations or medical devices or anywhere infectedmaterial may have contacted.

[0025] Compositions and methods of the invention are most effective whenthe pH is not at or near 7.0 or neutral e.g. not in the range of morethan 5.0 or less than 9.0. Preferably the pH is maintained at about 4.0or less for acid conditions or about 10.0 or more for basic conditions.Although the compositions of the invention are effective at high (above100° C.) and low (below 10° C.) temperatures a remarkable feature of thecompositions is that they are effective at mild temperatures such as 10°C. to 80° C. Although some improvement in the ability to inactivateprions is observed at temperatures such as 65° C. the formulation areeffective at body temperature i.e. 37° C. or less. The variousconditions such as temperature, pH, time of exposure, and concentrationof active ingredient in the formulation can vary and are interactive tosome degree. For example, decreasing temperature will generally requireincreasing the time of exposure to obtain the effect of inactivatinginfectivity.

[0026] A remarkable feature of the invention is that the inactivation ofprions generally requires an extremely harsh treatment. For example,conventional treatments include dry autoclaving at 132° C. for 4.5hours, liquid autoclaving for 4.5 hours at 132° C. in the presence of 1NNaOH, or incineration of the material at 1600° C. Since these harshconditions are difficult to carry out, time consuming and/or destructiveof materials the present invention provides a substantial advantage byinactivating prion infectivity at relatively mild conditions.

[0027] An antiseptic composition is disclosed which is comprised of afirst agent that maintains the pH of the composition at about 5.0 orless, preferably about 4.0 or less; and a second agent characterized byits ability to destroy prion infectivity in the low pH environmentcreated by the first component. The first agent may be any well knownacid present in an aqueous solution at sufficient molarity so as toreduce the pH of the antiseptic composition to about 5.0 or less, andpreferably about 4.0 or less and maintain the pH at that low level whenthe antiseptic composition is applied to an object or mixed with amaterial to be treated. The second agent or active agent ischaracterized by its ability to inactivate prions (destroy infectivity)when held in the acid environment for as little as two hours or less.The advantage of the composition is that it will not damage existingequipment or material because it is not applied under harsh conditions.

[0028] The composition of the invention will vary due to the largenumber of different acids and inactivating agents that can be used. Astemperature is increased and pH is lowered the inactivation occurs morerapidly. Compositions of the invention preferably inactivate prions inabout 2 hours or less at a pH of about 4.0 or less at temperatures ofabout 4° C. to 40° C. However, low temperatures (e.g., 0° C. or higher)and high temperatures (e.g., 100° C. or less) can be used, as can longertime periods. The inactivation can occur more quickly (e.g., in oneminute or less) and can be carried out at lower pH levels (e.g., 3.0 orless) and a higher temperatures (e.g., greater than 40° C.). An aspectof the invention is that the denaturing conditions are very mildcompared to the presently used methods to remove prions for example,from reusable medical equipment.

[0029] The inactivating component, or active agent, is best describedfunctionally, as those skilled in the art reading this disclosure willcontemplate other agents which could be used in a low pH composition toinactivate prions when the basic concepts and specific examples of theinvention are described. Some general classes of compounds useful as theactive agent include protein denaturants, inorganic salts; organicsolvents, detergents and dendrimers.

[0030] Compositions are disclosed that, when added to food products,will inactivate any prions present and prevent infection. Thecompositions may also be used on livestock feed, to disinfect any prionsin the livestock.

[0031] The antiseptic compositions of the invention can be combined withconventional antibacterial and antiviral agents in aqueous or alcoholsolutions to produce disinfecting agents or surgical scrubs. Branchedpolycations for use in the invention include, but are not limited to,polyamidoamide (PAMAM), polypropyleneimine (PPI), and polyethyleneimine(PEI) dendrimers, poly(4′-aza-4′-methylheptamethylene D-glucaramide),polyamidoamines and suitable fragments and/or variants of thesecompounds. Although polycationic dendrimers can be used as the activeagent in antiseptic formulations of the invention there are other morepreferred compounds that inactivate prions in an acid environment. Theessence of the invention is that a wide range of different types ofcompounds will render prions non-infectious in a relatively short periodof time (e.g., 2 hours or less) when maintained at a pH of 4.0 or lessat moderate temperatures, e.g., 4° C. to 37° C. In some cases, thecommercial value of the invention is decreased if the composition doesnot accomplish its intended purpose in a short time period (e.g., lessthan 10 minutes at about room temperature (20+5° C.)).

[0032] An overall aspect of the invention is an antiseptic compositioncomprising an aqueous solvent, an acid capable of maintaining thecomposition at a preferred pH of 4.0 or less and an active componentwhich at a low pH renders infectious prions non-infectious.

[0033] Another aspect of the invention is the use of a wide range ofprotein denaturants at low pH to inactivate prions.

[0034] An advantage of the invention is that proteins such as prions canbe rendered non-infectious without the need for extreme physicalconditions, such as exposure to heat over long periods of time, e.g.,without the need for an exposure of 1-10 hours at 100′-200° C.

[0035] Another feature of the invention is that compositions can beuseful while containing only very low concentrations of theprion-inactivating component such as SDS or polycationic dendrimers,e.g., 1% to 0.001%.

[0036] A further advantage of the invention is that conformationallyaltered protein such as prions can be rendered non-infectious with amethod which need only consist of applying an active component such asSDS or a polycationic dendrimer preferably held at a pH of 4.5 or less.

[0037] An important aspect of the invention is an assay whereby multiplecompounds can be quickly and easily tested for their ability to destroythe infectious character of prions while the compound and the prions areheld in a low pH environment.

[0038] Another aspect of the invention is the use of the claimedcompounds in livestock feed. This can prevent the transmission of prionsfrom livestock to humans by eliminating prions from the livestock beforeslaughter.

[0039] A further aspect of the invention is the use of the claimedcompounds to treat organs and tissues prior to transplantation, toeliminate any prions that may be present in the tissue that is to betransplanted.

[0040] A further aspect of the invention is the use of the claimedcompounds to treat donated blood or blood products before transfusion.

[0041] A further aspect of the invention is to pre-treat suture wirewith a composition of the invention before using on a patient.

[0042] These and other aspects, advantages, and features of theinvention will become apparent to those persons skilled in the art uponreading the details of the compounds, and methods more fully describedbelow.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] Before the present methods, objects and compositions aredescribed, it is to be understood that this invention is not limited tothe particular steps, devices or components described and, as such, mayof course vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to be limiting, since the scope of the present inventionwill be limited only by the appended claims.

[0044] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

[0045] The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates, which may need to be independently confirmed.

[0046] Definitions

[0047] The term “acid” is used to describe any compound or group ofcompounds that has one or more characteristics of (a) sour taste; (b)turns litmus dye red; (c) reacts with certain metals to form a salt; (d)reacts with certain bases or alkalines to form a salt. An acid compriseshydrogen and in water undergoes ionization so that H₃O⁺ ions are formed,also written as H⁺ and referred to as hydronium ions or simply hydrogenions. Weak acids such as acetic acid or carbonic acid may be used as maystrong acids such as hydrochloric acid, nitric acid and sulfuric acid(HCl, H₂SO₄, H₂NO₃). In compositions of the invention the acid ispreferably present in a concentration so as to obtain a pH of about 5 orless, more preferably about 4 or less and still more preferably 3.5±1.The acid component of the antiseptic composition must be present in aconcentration (molarity) to keep the composition in the desired pHrange. The concentration (molarity) or the acid used will vary somewhatwith the particular acid used, the solvent used (water or alcohol) andother factors such as temperature and pressure. The acid component ispreferably present in sufficient molarity and is of such type that whenthe antiseptic composition of the invention is put into use (e.g., mixedwith a sample to be disinfected) the composition remains within thepreferred pH range. Thus, stronger and/or more concentrated forms ofacids are preferred when the composition is to be used on or in asituation where the composition will be significantly diluted and/orcontact a high pH (i.e., very basic) component.

[0048] The terms “active component,” “active agent,” “inactivatingagent” and the like are used interchangeably herein to describe acompound or group of compounds which when combined in the “acid”component of the invention in the antiseptic composition will render aconformationally altered protein non-infectious. Preferably the activecomponent is within an environment of a pH of about 5 or less,preferably 4 or less and in a low concentration e.g., less than 5% byvolume of the composition, and with inactive prions or otherconformationally altered proteins in two hours or less, at a temperatureof 4° C. to 37° C. Active components can be determined using an assay ofthe invention whereby different compounds are tested for their abilityto destroy infectivity. Preferred compounds that act as an activecomponent for prions include SDS, urea, and a wide range of proteindenaturants including guanidine and thiocynate as well as variousbranched polycations. Some non-limiting examples of compounds whichcould be used as the active component include the following:

[0049] 1) Conventional protein denaturants including:

[0050] a) urea;

[0051] b) guanidine;

[0052] c) guanidine hydrochloride;

[0053] d) beta-mercaptoethanol;

[0054] e) dithiothreitol (DTT); and

[0055] f) chaotropes.

[0056] 2) Inorganic salts including:

[0057] a) lithium bromide;

[0058] b) thiocyanate;

[0059] c) potassium thiocyanate;

[0060] d) sodium iodide;

[0061] e) ammonium chloride, EDTA (metal chelator);

[0062] f) lithium ion and salts thereof; and

[0063] g) formic acid and salts thereof.

[0064] 3) Organic solvents including:

[0065] a) formamide;

[0066] b) dimethylformamide;

[0067] c) dichloro- and trichloroacetic acids and their salts; and

[0068] d) trifluroethanolamine (TFE).

[0069] 4) Detergents including:

[0070] a) sodium dodecyl sulfate (SDS) (also known as lauryl sulfate,sodium salt—other salts are also useful including lithium and potassiumsalts;

[0071] b) sodium cholate;

[0072] c) sodium deoxycholate;

[0073] d) octylglucoside;

[0074] e) dodecyldimethylamine oxide;

[0075] f) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate(CHAPS);

[0076] g) dodecyltriethylammonium bromide (DTAB);

[0077] h) cetyltrimethylammonium bromide (CTAB);

[0078] i) polyoxyethylene-p-isooctylphenyl ether (e.g., Triton X-20,Triton X-100, Triton X-114);

[0079] j) alkyl sulfate; and

[0080] k) alkyl sulfonate.

[0081] 5) Branched Polycations including:

[0082] a) polyamidoamide (PAMAM) dendrimers;

[0083] b) polypropyleneimine (PPI) dendrimers;

[0084] c) polyethyleneimine (PEI) dendrimers;

[0085] d) poly (4′-aza-4′-methylheptamethylene D-glycaramide);

[0086] e) polyamidoamines; and

[0087] f) fragments and variants of any of a-e.

[0088] Further information of compounds and conditions effecting proteinconformation can be found in Voet et al., Biochemistry, pp. 180-280(1990); Scopes, R. K., Protein Purification: Principles and Practice,pp. 57-71 (1987); and Deutscher, M. P., Guide to Protein Purification,pp. 240-241 (1990).

[0089] The term “detergent” is used to mean any substance that reducesthe surface tension of water. Examples of detergents are provided aboveas possible active components. The detergent may be a surface activeagent which concentrates at oil-water interfaces, exerts emulsifyingaction and thereby aids in removing soils e.g., common sodium soaps offatty acids. A detergent may be anionic, cationic, or monionic dependingon their mode of chemical action. Detergents include linear alkylsulfonates (LAS) often aided by “builders.” A LAS is preferably an alkylbenzene sulfonate ABS that is readily decomposed by microorganisms(biodegradable). The LAS is generally a straight chain alkyl comprising10 to 30 carbon atoms. The detergent may be in a liquid or a solid form.

[0090] The terms “prion,” “prion protein,” “infectious protein,”“PrP^(Sc) protein” and the like are used interchangeably herein to referto the infectious PrP^(Sc) form of a PrP protein, and is a contractionof the words “protein” and “infection.” Particles are comprised largely,if not exclusively, of PrP^(Sc) molecules encoded by a PrP gene. Prionsare distinct from bacteria, viruses and viroids. Known prions infectanimals to cause scrapie, a transmissible, degenerative disease of thenervous system of sheep and goats, as well as bovine spongiformencephalopathy (BSE), or “mad cow disease,” and feline spongiformencephalopathy of cats. Four prion diseases known to affect humans are:(1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3)Gerstmann-Sträussler-Scheinker Disease (GSS), and (4) fatal insomnia(FI). As used herein “prion” includes all forms of prions causing all orany of these diseases or others in any animals used; and in particularin humans and domesticated farm animals.

[0091] The term “conformationally altered protein” is used here todescribe any protein which has a three dimensional conformationassociated with a disease. The conformationally altered protein maycause the disease, be a factor in a symptom of the disease or appear asa result of other factors. The conformationally altered protein appearsin another conformation that has the same amino acid sequence. Ingeneral, the conformationally altered protein formed is “constricted” inconformation as compared to the other “relaxed” conformation, which isnot associated with disease. Those skilled in the art reading thisdisclosure will recognize the applicability of the antisepticcomposition of the invention to other conformationally altered proteinseven though the invention is described in general as regards to prions.

[0092] The following is a non-limiting list of diseases with associatedproteins, which assemble two or more different conformations, wherein atleast one conformation is an example of a conformationally alteredprotein. Disease Insoluble Proteins Alzheimer's Disease APP, Aβ peptide,α1-antichymotrypsin, tau, non-Aβ component, presenillin 1, presenillin2, apoE Prion diseases, PrP^(Sc) Creutzfeldt-Jakob disease, scrapie andbovine spongiform encephalopathy ALS SOD and neurofilament Pick'sdisease Pick body Parkinson's disease α-synuclein in Lewy bodiesFrontotemporal dementia tau in fibrils Diabetes Type II Amylin Multiplemyeloma- IgGL-chain plasma cell dyscrasias Familial amyloidoticTransthyretin polyneuropathy Medullary carcinoma Procalcitonin ofthyroid Chronic renal failure β₂-microglobulin Congestive heart failureAtrial natriuretic factor Senile cardiac and Transthyretin systemicamyloidosis Chronic inflammation Serum amyloid A Atherosclerosis ApoA1Familial amyloidosis Gelsolin Huntington's disease Huntingtin

[0093] The terms “sterilizing,” “making sterile” and the like are usedhere to mean rendering something non-infectious or rendering somethingincapable of causing a disease. Specifically, it refers to rendering aprotein non-infectious or incapable of causing a disease or the symptomsof a disease. Still more specifically, it refers to rendering aconformationally altered protein (e.g., PrP^(Sc) known as prions)incapable of causing a disease or the symptoms of a disease.

[0094] By “effective dose” or “amount effective” is meant an amount of acompound sufficient to provide the desired sterilizing result. This willvary depending on factors such as (1) the active agent used, (2) the pHof the antiseptic composition, (3) the type of object or material beingsterilized, and (4) the amount or concentration of infectious proteinswhich might be present. Polycations of the invention or morespecifically polycationic dendrimer compounds of the invention could bemixed with a material in an amount in a range 1 to 500 μg of dendrimerper ml or mg of material being sterilized. The concentration issufficient if the resulting composition is effective in decreasing(preferably eliminating) the infectivity of conformationally alteredproteins such that the treated material over time would not result ininfection. Because (1) some materials will have higher concentrations ofaltered protein than others; (2) some materials are contacted morefrequently than others; and (3) individual proteins have differentdegrees of infectivity, the effective dose or concentration range neededto sterilize can vary considerably. It is also pointed out that the doseneeded to treat an amount of material may vary somewhat based on the pHthe treatment is carried out at and the amount of time the compound ismaintained in contact with the material at the desired low pH (e.g., 4.5or less) level and the surrounding temperature and pressure.

[0095] The term “LD₅₀” as used herein is the dose of an active substancethat will result in 50 percent lethality in all treated experimentalanimals. Although this usually refers to invasive administration, suchas oral, parenteral, and the like, it may also apply to toxicity usingless invasive methods of administration, such as topical applications ofthe active substance.

[0096] The term “amine-terminated” includes primary, secondary andtertiary amines.

[0097] The terms “PrP protein,” “PrP” and like are used interchangeablyherein and shall mean both the infectious particle form PrP^(Sc) knownto cause diseases (spongiform encephalopathies) in humans and animalsand the noninfectious form PrP^(C) which, under appropriate conditionsis converted to the infectious PrP^(Sc) form.

[0098] The term “PrP gene” is used herein to describe genetic materialwhich expresses proteins including known polymorphisms and pathogenicmutations. The term “PrP gene” refers generally to any gene of anyspecies which encodes any form of a prion protein. Some commonly knownPrP sequences are described in Gabriel et al., Proc. Natl. Acad. Sci.USA 89:9097-9101 (1992) and U.S. Pat. No. 5,565,186, incorporated hereinby reference to disclose and describe such sequences. The PrP gene canbe from any animal, including the “host” and “test” animals describedherein and any and all polymorphisms and mutations thereof, it beingrecognized that the terms include other such PrP genes that are yet tobe discovered. The protein expressed by such a gene can assume either aPrP^(C) (non-disease) or PrP^(Sc) (disease) form.

[0099] The terms “standardized prion preparation,” “prion preparation,”“preparation” and the like are used interchangeably herein to describe acomposition (e.g., brain homogenate) obtained from the brain tissue ofmammals that exhibit signs of prion disease: the mammal may: (1) includea transgene as described herein; (2) have and ablated endogenous prionprotein gene; (3) have a high number of prion protein gene from agenetically diverse species; and/or (4) be a hybrid with an ablatedendogenous prion protein gene and a prion protein gene from agenetically diverse species. Different combinations of 1-4 are possible,e.g., (1) and (2). The mammals from which standardized prionpreparations are obtained exhibit clinical signs of CNS dysfunction as aresult of inoculation with prions and/or due to developing the diseaseof their genetically modified make up, e.g., high copy number of prionprotein genes. Standardized prion preparations and methods of makingsuch are described and disclosed in U.S. Pat. No. 5,908,969 issued Jun.1, 1999 and application Ser. No. 09/199,523 filed Nov. 25, 1998.

[0100] By “organ” is meant a differentiated structure consisting ofcells and tissues and performing some specific function in an animal.

[0101] By “tissue” is meant a group of cells similar to each other,along with their associated intercellular substances, which perform thesame function within a multicellular organism. Major tissue typesinclude epithelial, connective, skeletal, muscular, glandular, andnervous tissues.

[0102] The term “blood product” includes the red blood cells, whiteblood cells, serum or plasma separated from the blood.

[0103] The term “bodily fluid” encompasses blood, blood products, lymph,saliva, spinal fluid, semen, or any other fluid that originates in thebody.

[0104] Abbreviations used herein include:

[0105] CNS for central nervous system;

[0106] BSE for bovine spongiform encephalopathy;

[0107] CJD for Creutzfeldt-Jakob Disease;

[0108] FFI for fatal familial insomnia;

[0109] GSS for Gerstmann-Sträussler-Scheinker Disease;

[0110] AD for Alzheimer's disease;

[0111] CAA for cerebral amyloid angiopathy;

[0112] Hu for human;

[0113] HuPrP for human prion protein;

[0114] Mo for mouse;

[0115] MoPrP for mouse prion protein;

[0116] SHa for a Syrian hamster;

[0117] SHaPrP for a Syrian hamster prion protein;

[0118] PAMAM for polyamidoamide dendrimers;

[0119] PEI for polyethyleneimine;

[0120] PK for proteinase K;

[0121] PPI for polypropyleneimine;

[0122] PrP^(Sc) for the scrapie isoform of the prion protein;

[0123] PrP^(C) for the cellular contained common, normal isoform of theprion protein;

[0124] PrP 27-30 or PrP^(Sc) 27-30 for the treatment or proteaseresistant form of PrP^(Sc);

[0125] MoPrP^(Sc) for the scrapie isoform of the mouse prion protein;

[0126] N2a for an established neuroblastoma cell line used in thepresent studies;

[0127] ScN2a for a chronically scrapie-infected neuroblastoma cell line;

[0128] ALS for amyotrophic lateral sclerosis;

[0129] HD for Huntington's disease;

[0130] FTD for frontotemporal dementia;

[0131] SDS for sodium dodecyl sulfate; and

[0132] SOD for superoxide dismutase.

[0133] General Aspects of the Invention

[0134] The invention encompasses a range of antiseptic compositions,methods of rendering conformationally altered proteins non-infectious,and assays for determining compounds, which may be used as activeagents. The composition is comprised of an acid component and an activecomponent, although a single compound could serve the function of bothcomponents. The composition preferably comprises a solvent carriercomponent that is generally alcohol or aqueous based. The acid componentis characterized by maintaining the pH of the composition at 5.0 or lessand preferably at 4.0 or less when in use. The active component ischaracterized by rendering infectious proteins non-infectious.Preferably, the active component in the low pH environment of thecomposition renders infectious proteins non-infectious in two hours orless at a temperature of 40° C. or less.

[0135] Suitable acid components include a non-toxic weak acid such asacetic acid having dissolved therein an active component such as abranched polycation. Compositions of the invention may be in the form ofaqueous or alcohol solutions, which are comprised of a branchedpolycation, an antibacterial, an antifungal and an antiviral compound.The antiseptic compositions are coated on, mixed with, injected into orotherwise brought into contact with a material to be sterilized. Thecomposition is applied in a manner so that the branched polycation ismaintained at a low pH (e.g., 5 or less, and preferably 3.5±1) in anamount of 1 μg or more polycation per ml or mg of material to besterilized. The composition is maintained in the desired pH range at atemperature of 4° C. to 37° C., for a sufficient period of time (e.g.,preferably about 2 hours or less) to cause conformationally alteredprotein present on or in the material to be destroyed (e.g., hydrolyzed)or rendered non-infective. Preferred compositions of the invention areuseful in cleaning and sterilizing and may be comprised of an activeagent such as SDS or polycationic dendrimers, a detergent, and an acidcomponent providing a pH less than 3.5.

[0136] Dendrimer Compounds that Clear Prions

[0137] Dendrimers are branched compounds also known as “starburst” or“star” polymers due to a characteristic star-like structure (see FIG.1). Dendrimers of the invention are polymers with structures built fromAB_(n) monomers, with n 2 and preferably n=2 or 3. Such dendrimers arehighly branched and have three distinct structural features: 1) a core,2) multiple peripheral end-groups, and 3) branching units that link thetwo. Dendrimers may be cationic (full generation dendrimers) or anionic(half-generation dendrimers). For a review on the general synthesis,physical properties, and applications of dendrimers, see, e.g., Tomaliaet al., Angew Chem. Int. Ed. Engl. 29:138-175, (1990); and Y. Kim and C.Zimmerman, Curr. Opin. Chem. Biol. 2:733-7421 (1997).

[0138] In a preferred embodiment, sterilizing compositions of theinvention comprise a cationic dendrimer preferably dissolved in a low pHsolvent such as acetic acid. Examples of suitable dendrimers aredisclosed in U.S. Pat. Nos. 4,507,466, 4,558,120, 4,568,737, 4,587,329,4,631,337, 4,694,064, 4,713,975, 4,737,550, 4,871,779, and 4,857,599.Dendrimers typically have tertiary amines that have a pKa of 5.7. Thedendrimers can optionally be chemically or heat treated to remove someof the tertiary amines. Other suitable cations include polypropyleneimine, polyethyleneimine (PEI), which has tertiary amines with a pKa of5.9, and poly(4′-aza-4′-methylheptamethylene D-glucaramide), which hastertiary amines with a pKa of 6.0. The cationic dendrimer is preferablydissolved in the low pH solvent such as vinegar in a concentration of0.0001% or more, preferably 0.01% or more preferably about 1%.

[0139] Preferably, the dendrimers for use in the invention arepolyamidoamines (hereinafter “PAMAM”). PAMAM dendrimers are particularlybiocompatible, since polyamidoamine groups resemble peptide bonds ofproteins.

[0140] Dendrimers are prepared in tiers called generations (see,generations 0, 1 and 2 in FIG. 1) and therefore have specific molecularweights. The full generation PAMAM dendrimers have amine terminalgroups, and are cationic, whereas the half-generation dendrimers arecarboxyl terminated. Full generation PAMAM dendrimers are thus preferredfor use in the present invention. PAMAM dendrimers may be preparedhaving different molecular weights and have specific values as describedin Table 1 below for generations 0 through 10. TABLE 1 PAMAM DendrimersAnd Their Molecular Weights (Ethylene Diamine core, amine terminated)Generation Terminal Groups Mol. Wt. g/mol 0 4 517 1 8 1430 2 216 3256 332 6909 4 64 14,215 5 128 28,795 6 256 58,048 7 512 116,493 8 1024233,383 9 2048 467,162 10 4096 934,720

[0141] Table 1 shows the number of terminal amine groups for PAMAMdendrimers, generations 0 through 10, range from 4 to 4,096, withmolecular weights of from 517 to 934,720. PAMAM dendrimers are availablecommercially from Aldrich or Dendritech. Polyethyleneimine orpolypropylene dendrimers or quaternized forms of amine-terminateddendrimers may be prepared as described by Tomalia et al., Angew Chem.Int. Ed. Engl. 29:138-175 (1990).

[0142] Sterilizing Compositions

[0143] Examples provided herein show that various active compounds suchas SDS or highly-branched polycations, e.g., dendrimer compounds, at apH of 4.0 or less affect the extent and distribution of PrP^(Sc) proteindeposits in scrapie-infected cells. The presence of these activecompounds in a low pH environment and at relatively low, non-cytotoxiclevels results in a significant reduction in detectable PrP^(Sc) incells and brain homogenates. Thus, the present invention encompassescompositions for reducing, inhibiting, or otherwise mitigating thedegree of infectivity of a protein. A composition of the invention iscomprised of any compound capable of destroying conformationally alteredproteins when in a low pH environment (e.g., a detergent such as SDS ora polycationic dendrimer) in solution, suspension or mixture.

[0144] Sterilizing Formulations

[0145] Sterilizing compositions of the invention preferably contain theactive component in a concentration from 0.0001 to 10% of theformulation. The following methods and excipients are merely exemplaryand are in no way limiting.

[0146] In addition to including the active compound in the formulationit is important to maintain that compound in a low pH environment. Anynumber of known acids or mixtures of acids could be used with theinvention. Non-limiting examples of commercially available products,which could be supplemented with the cationic compounds, are describedbelow. In these formulations, the percentage amount of each ingredientcan vary. In general, a solvent ingredient (e.g., water or alcohol) ispresent in amounts of 40% to 100%. The other ingredients are present inan amount in a range of 1% to 60% and more generally 5% to 20%. In eachcase the polycationic compounds of the invention are added in amounts ofabout 0.01% to 5% and preferably 0.1% to 2% and more preferably about1%. The amount added is an amount needed to obtain the desired effect.Component wt % FORMULATION 1 Acid   90-99.99 Active component 0.01-10  FORMULATION 2 Acid   90-99.99 Protein denaturant 0.01-10   FORMULATION 3Acid   90-99.99 Inorganic salt 0.01-10   FORMULATION 4 Acid   90-99.99Organic solvent 0.01-10   FORMULATION 5 Acid   90-99.99 Detergent0.01-10   FORMULATION 6 Water 10-99 Acid  1-20 Active component0.01-10   of any of 1-5 FORMULATION 7 Water 10-98 Acid  1-20 Detergent 1-20 Polycationic dendrimer 0.01-5   FORMULATION 8 Water 10-98 Aceticacid  1-20 Linear alkyl 1-20 Sulfonate Polycationic 0.01-5   DendrimerFORMULATION 9 Water 10-99 Acetic acid  1-20 SDS 0.01-10   FORMULATION 10Water  1-98 Alcohol  0-98 Acid  1-20 Detergent  1-20 Polycationic0.1-5   Dendrimer FORMULATION 11 Water  1-99 Acid  1-20 Antibacterial0.1-5   Detergent  1-20 Polycationic 0.1-5   Dendrimer FORMULATION 12Water    3-98.889 Antimicrobial active 0.001-5    Agent Anionicsurfactant  1-80 Proton (H⁺) donating 0.1-12  Agent Polycationic0.01-5   dendrimer FORMULATION 13 Polycationic 0.5 dendrimer Ethanol74.0 Benzalkonium 0.2 Chloride CAE 0.02 Glycerine 1.0 Chain silicone 0.5Triglyceride 0.5 Lactic acid 10.0 Purified water 13.28 FORMULATION 14Polycationic 1.0 Dendrimer Ethanol 75.0 Benzalkonium 0.2 Chloride CAE0.02 Glycerine 1.0 Cyclic silicone 0.2 Triglyceride 0.3 Acetic Acid 20.0Purified water 2.28 FORMULATION 15 Polycationic 0.25 dendrimer Ethanol74.0 Chlorhexedine 0.75 Gluconate Benzalkonium 0.2 Chloride CAE 0.02Glycerine 2.0 Chain silicone 0.2 Cyclic silicone 0.2 Triglyceride 0.3Acetic Acid 20.0 Purified water 2.08 FORMULATION 16 Polycationic 0.1Dendrimer Ethanol 75.0 Chlorhexedine 0.9 Gluconate Benzalkonium 0.2Chloride CAE 0.02 Glycerine 1.0 Chain silicone 0.5 Cyclic silicone 0.5Triglyceride 0.3 Lactic acid 14.0 Purified water 7.98 FORMULATION 17Polycationic 0.01 dendrimer Ethanol 75.0 Benzalkonium 0.2 Chloride CAE0.02 Glycerine 2.0 Chain silicone 0.99 Cyclic silicone 2.0 Triglyceride3.0 Lactic acid 9 Purified water 7.78 FORMULATION 18 Polycationic 1Dendrimer Ethanol 75.0 Chlorhexedine 0.2 Gluconate Benzalkonium 0.2Chloride CAE 0.02 Glycerine 0.8 Chain silicone 0.2 Cyclic silicone 0.2Triglyceride .38 Acetic acid 10 Purified water 12 FORMULATION 19Polycationic 0.001 dendrimer Ethanol 75.99 Chlorhexedine 0.2 GluconateCAE 0.02 Glycerine 1.0 Chain silicone 0.2 Triglyceride 0.3 Lactic acid14 Purified water 8.28 FORMULATION 20 Polycationic 1 Dendrimer Ethanol75.0 Benzalkonium 0.2 Chloride CAE 0.02 1,3-butylene glycol 1.0Metylphenyl 0.2 polysiloxane Isopropyl myristate (IPM) 0.3 Purifiedwater 22.28 FORMULATION 21 Water  1-98 Alcohol  0-98 Acid  1-20 SDS 1-20 FORMULATION 22 Water  1-99 Acid  1-20 Antibacterial agent 0.1-5  Detergent  1-20 FORMULATION 23 Water    3-98.889 Antimicrobial active0.001-5    Agent Anionic surfactant  1-80 Proton (H⁺) donating 0.1-12 Agent SDS 0.01-5   FORMULATION 24 SDS 0.5 Ethanol 74.0 Benzalkonium 0.2Chloride CAE 0.02 Glycerine 1.0 Chain silicone 0.5 Triglyceride 0.5Lactic acid 10.0 Purified water 13.28 FORMULATION 25 Urea 1.0 Ethanol75.0 Benzalkonium 0.2 Chloride CAE 0.02 Glycerine 1.0 Cyclic silicone0.2 Triglyceride 0.3 Acetic Acid 20.0 Purified water 2.28 FORMULATION 26Guanidine 0.25 hydrochloride Ethanol 74.0 Chlorhexedine 0.75 GluconateBenzalkonium 0.2 Chloride CAE 0.02 Glycerine 2.0 Chain silicone 0.2Cyclic silicone 0.2 Triglyceride 0.3 Acetic Acid 20.0 Purified water2.08 FORMULATION 27 Thiocynate 0.1 Ethanol 75.0 Chlorhexedine 0.9Gluconate Benzalkonium 0.2 Chloride CAE 0.02 Glycerine 1.0 Chainsilicone 0.5 Cyclic silicone 0.5 Triglyceride 0.3 Lactic acid 14.0Purified water 7.98 FORMULATION 28 Sodium deoxycholate 0.01 Ethanol 75.0Benzalkonium 0.2 Chloride CAE 0.02 Glycerine 2.0 Chain silicone 0.99Cyclic silicone 2.0 Triglyceride 3.0 Lactic acid 9 Purified water 7.78FORMULATION 29 SDS 1 Ethanol 75.0 Chlorhexedine 0.2 GluconateBenzalkonium 0.2 Chloride CAE 0.02 Glycerine 0.8 Chain silicone 0.2Cyclic silicone 0.2 Triglyceride .38 Acetic acid 10 Purified water 12FORMULATION 30 SDS 0.001 Ethanol 75.99 Chlorhexedine 0.2 Gluconate CAE0.02 Glycerine 1.0 Chain silicone 0.2 Triglyceride 0.3 Lactic acid 14Purified water 8.28 FORMULATION 31 Sodium Acetate 10% pH 4.0 ± 1 SDS 4%Water 86% FORMULATION 32 SDS 4% Peracetic acid  0.1-10%  Water  86-95.9% FORMULATION 33 SDS 4% Glycerine 10% Water 86% FORMULATION 34SDS   1-20% Acid pH 4.0 ± 1   1-20% Water  60-98% FORMULATION 35 SDS  1-20% Base pH 10.0 ± 1   1-20% Water  60-98% FORMULATION 36 SDS 1Ethanol 75.0 Benzalkonium chloride 0.2 CAE 0.02 1,3-butylene glycol 1.0Metylphenyl 0.2 polysiloxane Isopropyl Myristate (IPM) 0.3 Purifiedwater 22.28 FORMULATION 37 Base   90-99.99 Active component 0.01-10  FORMULATION 38 Base and solvent   90-99.99 Salt of alkyl sulfate0.01-10   FORMULATION 39 Base and water   90-99.99 Sodium dodecylsulfate 0.01-10   FORMULATION 40 Base and alcohol   90-99.99 Sodiumdodecyl sulfate 0.01-10   FORMULATION 41 Na OH and water   90-99.99Sodium dodecyl sulfate 0.01-10  

[0147] Certain compositions are more economical and efficient to produceand are therefore more likely to be commercial compositions. An exampleof such would include sodium dodecyl sulfate in combination with aceticacid and/or parasitic acid. Further, because the ability of formulationsof the invention to be effective in inactivating prions somewhat pHdependent a product could be sold in combination with a pH indicator toensure that the appropriate pH had been reached. Preferably, theindicator would be an indicator which would only show acid conditionswhen the pH was at or below a desired level or when the pH was at orabove a desired level. Thus, a modified litmus paper could be providedwhich, for example, indicated red or pink only when the pH was at 4 orless and indicated blue or violet only when the pH was at 10 or higher.Thus, by using the indicator the user could be assured that the properpH had been obtained for the formulation to be effective in activatingthe infectivity of prions.

[0148] By using the disclosure provided here and other information suchas taught in U.S. Pat. Nos. 5,767,054; 6,007,831; 5,830,488; 5,968,539;5,416,075; 5,296,158; and patents and publications cited therein, thoseskilled in the art can produce countless other formulations of theinvention. Further, the formulations are preferably adjusted to have apH of less than 4.0, and such formulations can be used as described insuch publications and can be packaged in any suitable container ordispenser device, e.g., taught in U.S. Pat. No. 5,992,698. The pH can belowered with any acid, e.g., HCl, H₂SO₄, H₂NO₃ peracetic acid, etc., andcan be used as the acid component in the formulae provided above.

[0149] Example 17, discussed below, shows that compounds such as SDS areeffective in denaturing PrP^(Sc) not only at a low pH (e.g., 5 or less)but also are effective at a high pH (e.g., 9 or more), while generallynot effective at a pH of about 7.0±1. The pH of the formulation can beadjusted to obtain desired results in each particular use. For example,a very high or very low pH may be best for inactivating PrP^(Sc); theseextreme pH's may be undesirable in some situations due to theircorrosive effects. Thus, a preferred pH is one that inactivates PrP^(Sc)and has the least possible adverse effects for the intended use. In manysituations a preferred pH for an SDS formulation is about 4.0±1 or about10.0±1.

[0150] Formulations of the invention used with a cell culture have theadvantage that they are non-toxic. For example, parenteraladministration of a solution of the formulations of the invention ispreferably nontoxic at a dosage of 0.1 mg/mouse, which is an LD₅₀ ofless than one at 40 mg/kg. Various nutrient formulations and/orinjectable formulations of the type known to those skilled in the artcan be used to prepare formulations for treating cell cultures.

[0151] Those skilled in the art will understand that in some situationsit may be desirable to further reduce the pH environment to obtain thedesired results. This can be accomplished by adding any desired acid. Ifdesired, the pH can be raised to a normal level after treatment iscomplete, i.e., after a sufficient amount of any conformationallyaltered protein present is destroyed.

[0152] Compounds effective in sterilizing compositions containingconformationally altered proteins are determined via a cell cultureassay and an organ homogenate assay each of which is described below indetail.

[0153] Livestock Feed

[0154] An important application of the invention is a composition thatrenders prions non-infectious and/or prevents prion formation and/oraids in the denaturation of prions from a mammal when combined with alivestock feed. In particular, a composition of the invention iscombined with a livestock feed, which is derived from an animal sourcesuch as meat or bone meal and more particularly animal material, whichincludes ground material from the central nervous system from anotheranimal. However, the compounds of the invention can be combined withplant-derived materials used in livestock feed in order to render prionsnon-infectious and/or prevent or treat prion infections in the animaleating the feed. More particularly, the compounds of the presentinvention can be added to livestock feed or feedstuffs used to feed anytype of livestock. The feedstuff compositions disclosed herein areintended to provide nutritional requirements of a variety of animals,including cattle, poultry, swine, sheep, goats, other monogastric orruminant livestock. The composition generally varies according to thetype of animals to which the feedstuff will be given.

[0155] Examples of various animal feedstuff components can be found inU.S. Pat. Nos. 6,207,217, 6,203,843, 5,786,007, 4,225,621, 4,161,543 and4,062,988, the disclosures of which are herein incorporated byreference.

[0156] Generally, when the term “feedstuff” is used with respect to thepresent invention, the term comprises all types of plant and animalcomponents. Specifically, “feedstuff” includes organic components suchas proteins, crude fiber, acid detergent fiber, neutral detergent fiber,vitamins and minerals. Typical compositions of feedstuffs for livestockinclude, but are not limited to, the following components: alfalfas,ammonium sulfate, barleys, beet pulps, blood meal, bluestem grass,brewers grains and yeast, brome grass, calcium carbonate, canary grass,carrot pulp, roots and tops, cattle manure, cheatgrass, clovers, coffeegrounds, corn and corn plants, cottonseed, defluorinated phosphate,diammonium phosphate, dicalcium phosphate, distillers grain barley,distillers grain corn, feathermeal hydrolyzed, garbage (municipal),grain screenings and grain dust, grape pomace, grass silage, hominyfeed, hop leaves, vines and spent hops, limestone, linseed meal, alltypes of hay including meadow hay, meat and bone meal (MBM), milo grain,mint slug silage, molasses beet, cane, citrus and wood, monoammoniumphosphate, mono-dicalcium phosphate, navy beans, all types of oatsincluding oat hay, oat silage, oat straw, oat grain, groats, oat meal,oat mill byproducts and oat hull, orange pulp, orchard grass, pea vines,peanut hulls, skins and meal, potato vine, potatoes and potato waste,poultry fat and poultry litter and manure, prairie hay, rapemealsolvent, rye straw and grain, safflower meal, sagebrush, sorghum stoverand silage, soybeans and soybean hull, sudangrass hay and silage,sunflower meal and hulls, timothy hay and silage, tomatoes, triticalesilage, urea, wheat bran, wheat grass, wheat grain, wheat shorts andwheat straw.

[0157] Further, the above feedstuff components are set forth above,serve merely as examples and are not intended to be comprehensive orlimiting. As such, suitable feedstuffs for the present invention maycomprise additional components not provided in the list above.

[0158] Of the above listed feed components, meat and bone meal (MBM)stands out as one the richest sources of energy and minerals. Typically,the crude protein content of MBM is about 50%. Hamilton, C. R., “Meatand Bone Meal,” Esteem Products. Vol. 1(1). MBM is thus one of the mostefficient feed components. MBM is produced as a by-product from theremoval of fat from animal tissues through rendering. The renderingprocess produces a finely ground, dry residue of animal by-productspressure cooked and stabilized by high temperature steam in closedtanks. The fat can be skimmed off and the solid residue is pressed toremove as much of the fat and water as possible. As defined andregulated by the Association of American Feed Control Officials (AAFCO),MBM is the rendered product from mammal tissues, including bone,exclusive of any added blood, hair, hoof, horn, hide trimmings, manure,stomach and rumen contents, except in such amounts as may occurunavoidably in good processing practices. As such, neuronal tissues areincluded in MBM products. (See, “The BSE Inquiry” § 9.15 athttp://www.bse.org.uk/report/volume7/chapteh2.htm).

[0159] The invention comprises feedstuff as defined herein, incombination with a composition that inhibits prion formation. Acomposition of the invention is added to feedstuff and fed to an animal,and in particular to domesticated livestock farm animals such as cows,pigs, sheep, goats, horses, chickens etc. The active component is addedin an amount sufficient to “treat” the animal. The amount will varybased on factors such as the type of animal and its size. In general,dosing is such that the animal will receive about 10 mg to about 10,000mg/day/kg of weight of the animal.

[0160] Blood and Blood Products

[0161] A further aspect of the invention is the use of the claimedcompounds and methods to treat blood and blood products prior totransfer to a recipient. Application of the composition of the inventionrenders prions non-infectious and/or prevents prion formation and/oraids in the denaturation of prions from blood and blood products.Approximately 14 million blood donations occur every year in the UnitedStates. About 12 million units of whole blood are transfused annually.There have been chronic shortages of blood, partly because of increaseddemand from modern surgical techniques. For example, people who areundergoing aggressive cancer chemotherapy treatments require bloodtransfusions because their own body's ability to make blood cellsdiminishes. Premature infants may require blood transfusions to carryoxygen throughout their bodies. Medical treatments, such as organtransplants and cardiac bypass surgery, that require a large amount ofblood, were uncommon 20 years ago, yet today are routine. And the agingof the population means that more people live longer and are more likelyto need medical treatments that require safe blood and blood products.Last year the American Red Cross, which provides one-half of thenation's blood supply to patients throughout the country, collected morethan 6.3 million volunteer blood donations. However, the demand forblood has been increasing even faster than the supply.

[0162] Compounding the problem of low blood supply is the emergence ofCreutzfeldt-Jakob Disease. Many potential blood donors are now excludedfrom donating because of the risk of prior exposure to the disease.There is some evidence to date that CJD can be transmitted through bloodtransfusions, however no cases have been found in the United States. Theevidence is not definitive, however, and questions remain as to whetheror not there are prions in the U.S. blood supply. In response to thisthreat, the U.S. Food and Drug Administration is implementing a ban ondonations from people who have spent more than ten years in France,Portugal, or Ireland since 1980. People who have spent more than sixmonths in Great Britain from 1980-1996 are already forbidden from givingblood in the United States, Canada, New Zealand, and Australia. Atpresent, there is no commercial diagnostic test for detecting prions inthe blood of humans or other animals. Humans may be infected with prionsfor five to twenty years before symptoms appear, which means that theU.S. blood supply could already be tainted with prions.

[0163] Further restrictions on donors will exacerbate an alreadycritical shortage of donated blood in the United States. Because of thepossibility of transferring prions via blood and plasma transfusions,the instant invention is useful in preventing the transfer of prions inthe blood supply by disinfecting the existing supply of blood and bloodproducts. This would decontaminate any prions in the existing bloodsupply, and would also allow more people to donate blood who are unableto do so at the present time because of the current restrictions.

[0164] Suitable procedures for removing prions from blood may be foundin Pruisner et al., U.S. Pat. No. 6,221,614. The procedure generallyencompasses exposure of the blood or blood product to a complexing agentthat usually is immobilized on a solid surface. Prions present in theblood or blood product will complex to the complexing agent, which isthen removed from the sample, and the filtered blood or blood product isthen free of any prion infection.

[0165] The blood should be treated preferably at room temperature (15°C. to 30° C.). The solution should have a pH of about 6.4 to 8.4,preferably 7.4, and should not contain excess magnesium or calcium. Asample is then exposed to a complexing agent, which is immobilized on asolid surface or otherwise provided in a manner allowing separation ofthe prion-bound complexing agent from the sample. The complexing agentforms a complex with, or somehow binds preferentially with, orexclusively to any constricted form (generally a pathogenic PrP^(Sc)form) of the protein present in the sample, thus effectivelyimmobilizing any PrP^(Sc) present in the sample to the solid surfaceupon exposure of the sample to the immobilized complexing agent.

[0166] In one embodiment, a chemical agent such as a heteropoly acid(e.g. PTA), or preferably a metallic salt thereof (NaPTA) is immobilizedto a solid surface such as a membrane filter, a magnetic bead, and thelike. The sample is subjected to a complexing agent over a period oftime sufficient to allow substantially all the PrP in the sample tocomplex with the PTA. For example, the sample could be incubated atabout 30° C. to 45° C. (preferably 37° C.) over a period of from about 1to 16 hours. The complexing agent forms a complex with the PrP^(Sc).What is important is that complex formed can be separated away from therest of the sample by some means, e.g., filtration, use of magneticfield, sedimentation and the like.

[0167] The present invention may be used to treat a biological samplewherein the PrP^(Sc) or other pathogenic protein is substantiallyremoved from the sample, and preferably to levels at which the PrP^(Sc)is undetectable by conventional means. Methods of removal of thePrP^(Sc) will aid in preventing transmission of PrP^(Sc)-mediateddisorders by providing biological samples that are substantially freefrom infectious levels of prions, i.e., “prion free.” Further, anyprions not removed from the sample after treatment with a composition ofthe invention are rendered non-infectious.

[0168] It is not possible to precisely determine the conditions underwhich the infectivity of an infectious protein such as a prion can beeliminated or the conditions under which the pathogenic proteins beingtreated will be denatured. Such will vary depending on the particularactive component being utilized. However, an important aspect of theinvention is that the active component be able to eliminate infectivityor denature an infectious protein such as prions under relatively mildconditions. In general, these conditions include a pH of about 5 or lessat a temperature above 4° C. for a period of time of about 2 hours ormore. However, it is not necessary generally to raise the temperatureabove 100° C. and it is generally possible to inactivate infectivity ordenatured proteins in a range of 20 to 100° C. or more preferably 37° C.to 80° C. or still more preferably in a range of about 10° C. to 60° C.at a pH in a range of about 3 to 5 over a period of about 2 hours orless and more preferably 1 hour or less.

Complexing Agents

[0169] Compounds that are useful as complexing agents in the presentinvention include antibodies, enzymes, peptides, chemical species,binding molecules, etc. These complexing agents are used in a mannerthat allows binding and removal of prions from a biologial solution,while maintaining the essential elements of the biological materialintact, e.g. retention of cellular morphology and protein integrity.Such complexing agents may be used in whole blood, in blood componentssuch as plasma and platelets, and in other biological fluids, as will beapparent to one skilled in the art.

[0170] Chemical Agents

[0171] In one embodiment of the prion removal pre-treatment step of theinvention, the compound for removal of prions from a biological materialis a chemical agent that precipitates PrP^(Sc). One preferred class ofchemical agents for use as complexing agents in the present inventionare heteropoly acids and salts thereof. Heteropoly acids are fully orpartially protonated forms of oxyanions having at least one centralelement and at least one coordinating element. Heteropoly acids may havethe Keggin or Dawson structures.

[0172] A particular class of heteropoly acids is the protonated form ofheteropolymolybdates. These anions contain from 2 to 18 hexavalentmolybdenum atoms around one or more central atoms. About 36 differentelements have been identified as central atoms of theseheteropolymolybdates. These anions are all highly oxygenated. Examplesof heteropolymolybdates include [PMo₁₂ O₄₀]³, [As²Mo₁₈O₆₂]⁶, and[TeMo₆O₂₄]⁶, where the central atoms are P⁵⁺, As⁵⁺, and Te⁶⁺,respectively. A more detailed discussion of heteropolymolybdates isprovided in the Kirk-Othmer Encyclopedia of Chemical Technology, 3rded., 15, pp. 688-689 (1981).

[0173] Another class of heteropoly acids, which is analogous to theprotonated form of heteropolymolybdates, is the protonated form ofheteropolytungstates. In heteropolytungstates, the coordinating elementis tungsten instead of molybdenum. See, U.S. Pat. No. 4,376,219. Thecentral elements of these heteropoly acids may be selected from thegroup consisting of P, Si, B, Ge, As, Se, Ti, Zr, Mn, F, V, Ce, and Th.The coordinating elements of these heteropoly acids include Mo and/or W.Optional coordinating elements include V, Mn, Co, Ni, Cu, Zn, and Fe.The ratio of the number of the coordinating elements to the number ofcentral elements may be from 2.5 to 12, preferably from 9 to 12.Particular heteropoly acids, which are exemplified in U.S. Pat. No.4,376,219, include phosphotungstic acid, silicotungstic acid,10-tungsto-2-vanadophosphoric acid, 6-tungsto-6-molybdophosphoric acid,phosphomolybdic acid, silicomolybdic acid, germanotungstic acid,tungstofluoric acid, and 18-tungsto-2-phosphoric acid, as well as saltsof all or any of these acids, e.g., metal salts such as Na, K, Mg, andCa salts. A particular heteropoly acid for use in the present inventionis phosphotungstic acid, i.e., H₃ PW₁₂ O₄₀ and its metal saltsparticularly Na salts. Such complexing agents effectively bind toPrP^(Sc).

[0174] Such chemical agents may be used alone, in combination, or withother non-bioactive chemicals such as buffers and inert bindingchemicals. Heteropoly acids of the invention (e.g., PTA) are preferably,although not exclusively, used in a metallic salt form. The metallicsalt includes, but is not limited to, sodium, potassium, calcium and thelike.

[0175] The amount of heteropoly acid or salt thereof which is combinedand/or coated onto a support material is an amount sufficient tosignificantly remove PrP^(Sc) from the a biological fluid, andpreferably in an amount sufficient to remove PrP Sc to undetectablelevels or at least non-infectious levels. The weight ratio of heteropolyacid to support material may be, for example, from about 1:20 to about1:1. The heteropoly acid may be combined with the support material inany manner, which provides adequate dispersion of the heteropoly acid,thereby increasing the effective surface area of the heteropoly acid. Apreferred technique for combining these components is by impregnation ofthe support material with the heteropoly acid. The heteropoly acid mayalso be combined with the support material by an ion exchange technique.The impregnation technique may involve sorbing an aqueous solution ofthe heteropoly acid into the porous region of the support materialfollowed by drying to remove water and to leave behind supportedheteropoly acid. Other methods of immobilizing heteropoly acids or saltsthereof may be used to immobilize these complexing agents, as will beapparent to one skilled in the art upon reading this disclosure.

[0176] Biological Agents

[0177] In another embodiment, the complexing agent is a protein,peptide, or other biological moiety that selectively binds to PrP^(Sc).

[0178] In one embodiment, the complexing agents are peptides or othersmall molecules designed to selectively bind to prions. Preferably, thepeptides or small molecules are designed to preferentially bind toPrP^(Sc). By “preferentially bind” is meant that the peptide is designedto be at least 20 times or more, more preferably 50 times or more, morepreferably 100 times or more, and even more preferably 1000 times ormore likely to bind to PrP^(Sc) than to other proteins in the biologicalsolution. Peptides of the invention are preferably designed to bind tothe native form of PrP^(Sc), as opposed to the denatured form, since thebiological fluids generally contain PrP^(Sc) in native form. Peptidesmay be designed to maximize binding to PrP^(Sc) by designing thepeptides to areas of PrP^(Sc) that are more accessible to binding, ascan be predicted by one skilled in the art. Useful antibodies that bindPrP^(Sc) are disclosed and described in U.S. Pat. No. 5,846,533.Portions of these antibodies, which bind to PrP^(Sc) are peptides whichcan be bound to a support surface and used in the present invention.

[0179] Alternatively, peptides may be designed to bind selectively toPrP^(C) or to both PrP^(Sc) and PrP^(C). Although the PrP^(Sc) form ofthe PrP protein is the infectious form, removal of the normal cellularprion proteins can also effectively halt or slow the progression of aprion-mediated disorder.

[0180] The complexing agent of the invention may also be an antibodyselective for prions. This antibody may be directly immobilized or maybe bound to another component (e.g., a high density metal). Thatantibody may bind to PrP^(Sc), e.g., the antibody disclosed in U.S. Pat.No. 5,846,533. To remove PrP^(C) present in the sample, an antibody thatbinds selectively or exclusively to PrP^(C) may be used. Such anantibody is disclosed in U.S. Pat. No. 4,806,627, issued Feb. 21, 1989,disclosing monoclonal antibody 263K 3F4, produced by cell line ATCCHB9222 deposited on Oct. 8, 1986. The cell line producing the antibodycan be obtained from the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. 20852.

[0181] In general, scrapie infection fails to produce an immuneresponse, with host organisms being tolerant to PrP^(Sc) from the samespecies. Antibodies that bind to either PrP^(C) or PrP^(Sc) aredisclosed in U.S. Pat. No. 5,846,533. Any antibody binding to PrP^(C)and not to PrP^(Sc) can be used, and those skilled in the art cangenerate such using known procedures, e.g., see methods of producingphage display antibody libraries in U.S. Pat. No. 5,223,409. Polyclonalanti-PrP antibodies have though been raised in rabbits followingimmunization with large amounts of formic acid or SDS-denatured SHaPrP27-30. The antibodies were generated against formic acid- orSDS-denatured PrP 27-30 and are able to recognize native PrP^(C) andtreated or denatured PrP^(Sc) from both SHa and humans equally well, butdo not bind to MoPrP. Not surprisingly, the epitopes of these antibodieswere mapped to regions of the sequence containing amino acid differencesbetween SHa- and MoPrP (Rogers et al. (1993) Proc. Natl. Acad. Sci. USA90:3182-3186).

[0182] It is not entirely clear why many antibodies of the typedescribed in the above cited publications will bind to PrP^(C) andtreated or denatured PrP^(Sc) but not to native PrP^(Sc). Without beingbound to any particular theory, it is believed that such may take placebecause epitopes that are exposed when the protein is in the PrP^(C)conformation are unexposed or partially hidden in the PrP^(Sc)configuration—where the protein is relatively insoluble and morecompactly folded together.

[0183] For purposes of the invention, an indication that no bindingoccurs means that the equilibrium or affinity constant K_(a) is 10⁶l/mole or less. Further, binding will be recognized as existing when theK_(a) is at 10⁷ l/mole or greater, preferably 10⁸ l/mole or greater. Thebinding affinity of 10⁷ l/mole or more may be due to (1) a singlemonoclonal antibody (i.e., large numbers of one kind of antibodies) or(2) a plurality of different monoclonal antibodies (e.g., large numbersof each of five different monoclonal antibodies) or (3) large numbers ofpolyclonal antibodies. It is also possible to use combinations of(1)-(3). Selected preferred antibodies will bind at least 4-fold moreavidly to the treated or denatured PrP^(Sc) forms of the protein whencompared with their binding to the native conformation of PrP^(Sc). Thefour-fold differential in binding affinity may be accomplished by usingseveral different antibodies as per (1)-(3) above and as such some ofthe antibodies in a mixture could have less than a four-fold difference.

[0184] A variety of different methods may be used with one or moredifferent antibodies. Those of skill in the art will recognize thatantibodies may be labeled with known labels and used with currentlyavailable robotics, sandwich assays, electronic detectors, flowcytometry, and the like. Further, the antibodies may be bound to densercomponents directly or via other intermediates such as anti-antibodies.

Methods of Purification

[0185] The complexing agent used in an initial clean-up or pre-treatmentstep of the invention, may be used in a variety of purificationprocedures to effectively remove prions from a biological material. Anumber of methods for use in the present invention are summarized asfollows.

[0186] Affinity Chromatography

[0187] Affinity chromatography (AC) relies on the interaction of theprotein with an immobilized ligand. AC is predicated, in part, on theinteraction of ligands attached to chromatographic supports. Ahydrophobic ligand coupled to a matrix is variously referred to hereinas an AC support, AC gel or AC column. It is further appreciated thatthe strength of the interaction between the protein and the AC supportis not only a function of the proportion of non-polar to polar surfaceson the protein but by the distribution of the non-polar surfaces aswell.

[0188] A number of matrices may be employed in the preparation of ACcolumns. Preferably, such matrices are beads, and more preferablyspherical beads, which serve as a support surface for the complexingagent of the invention. Suggested materials for the matrices includeagarose, cross-linked dextran, polyhydroxyl ethyl methacrylate,polyacrylamide, cellulose, and derivatives or combinations thereof,preferably in the form of porous spheres. Cellulose acetate haspreviously been successfully used in devices for purification ofbiolological fluids, e.g., extracorporeal blood purification devices.Polyurethane is particularly blood compatible. Silica and itsderivatives are also especially useful as support material for use withheteropoly acids. See U.S. Pat. Nos. 5,475,178 and 5,366,945.

[0189] The preferred material for use in the methods of the presentinvention is agarose, a naturally occurring hydrophilic polymer. Abeaded gel with a porosity of from 90-96% is formed by varying thepercentage of agarose. The molecular weight of the gel ranges from 0.5million for 10% agarose to 20 million for 4% agarose. Particle diametersranging from 20 to 200 microns are commercially available. Themechanical strength of agarose beads can be increased by eitherincreasing the percentage of agarose or crosslinking the beads withepichlorohydrin or 2,3 dibromopropanol, using the method of J. Porath etal., J Chromat. 60:167 (1971). This allows a corresponding increase inthe maximum operating pressure (a fifty percent increase in agaroseleads to a two to four fold increase in the maximum operating pressure).

[0190] The criteria to determine the appropriate coupling method are:minimization of leakage of the complexing agent from the support,maintenance of the thermal stability of the compound, and retention ofthe optimum amount of complexing agent. The technique must also notcause deterioration in the support material or the production ofreactive groups on the support that would bind blood components in vivo.The complexing agent must also retain its activity over time.

[0191] Further factors which must be considered in optimizing theaffinity chromatography coupling method are: the extent of distributionof the coupling agent within the particles and/or columns; pH;temperature; the flow speed of the biological sample through the column;the size of the bound complexing agent; and/or the diameter and poresize of the particular support. Each of these conditions can beoptimized for a particular procedure, biological sample, and complexingagent as will be apparent to one skilled in the art.

[0192] The AC composition of the present invention can be containedwithin a filtration cartridge for easy use of the composition in abiological fluid purification process. When the column composition iscontained within a single cartridge, it can easily and conveniently bereplaced when the purifying capacity of the composition becomesexhausted. Alternatively, the cartridge may be an integral part of apurifying device, in which case the entire device is replaced once thefiltration composition has exhausted its efficacy. The support particleswith the complexing agent are placed within the cartridge, and thesolution to be reacted with the complexing agent is then circulatedthrough the cartridge. Commercially available units for dialysis, bloodexchange or oxygenation can be adapted for use as the purifying device.

[0193] Filtration Methods

[0194] Another method that may be used to remove prions from abiological sample involves filtration through a membrane. The membranemay have the prion complexing agent conjugated directly to the membrane,either on the side facing the biological fluid or more preferably on theside away from the biological fluid. Alternatively, the complexing agentmay be compartmentalized in an area behind the membrane, which isinaccessible to the larger components of the biological materials, e.g.,blood cells. In the latter example, the complexing agent can be bound toan insoluble matrix behind the membrane. The membrane for use in thepresent invention may be in planar form, in the form of one or morehollow fibers, and/or in the form of flat foils. See U.S. Pat. No.4,361,484.

[0195] Suitable materials for the membrane include regeneratedcellulose, cellulose acetate, non-woven acrylic copolymer, polysulphone,polyether sulphone, polyacrylonitrile, polyamide and the like. Thebiologically active material is immobilized in the pores and/or on thesurface of the side of the membrane that faces away from the biologicalfluid. Thereby the components, such as blood corpuscles, are preventedfrom contacting the active material. The pores of the membrane areusually of the magnitude of order of 0.01 to 0.8 microns, preferably0.15 to 0.45 microns. The polymer support must be stable under theconditions of its planned use, i.e., it should not be chemically orenzymatically degraded by blood. The support and immobilized complexingagent must be blood compatible, and the support should have good flowcharacteristics and low compressibility under clinical flow rates in therange of 150-250 ml/min.

[0196] Through the above construction of the microporous membrane, i.e.,asymmetric immobilizing of the prion complexing agent, the biologicalfluid need not be exposed to any following filtering for removingpossible remaining harmful residues. The separation as the removal ofthe substances can thereby be performed in one and the same step.

[0197] The microporous semipermeable membrane can be in the form ofindividual fibers, which are bundled and encapsulated within one and thesame casing, with an inlet and outlet for the biological fluid. The endsof the fibers are glued by means of a suitable binder to retain theindividual fibers essentially parallel within the casing. One end of thefibers or bundles of fibers is provided in communication with the inlet,while the opposite end is provided in communication with the outlet.

[0198] The biological material is pumped into the casing through theinlet and through the longitudinal void of the fibers and out of thecasing through the outlet. During the passage through the casing thefluid is exposed to the pressure variations, such that only apenetrating fraction is caused to flow in an alternating path throughthe fiber walls in each direction for contacting with the prioncomplexing material. The means for the realization of the pressurevariations may again be made up of an expansion chamber in communicationwith the space between the individual fibers and bundles of fibers,respectively. Any subsequent filtering of the biological material forthe removal of possible harmful residues is not needed, since thefiltering is automatically achieved through the passage of the fluidthrough the fiber walls.

[0199] The pressure variations may vary from −200 to +200 mmHg,preferably from −100 to +100 mmHg. The longer the diffusion distance forthe blood, for example if the prion complexing agent is bound to anunsoluble matrix behind the membrane, the higher compensating pressurevariations are required to achieve the desired separation effect. In acorresponding way the frequency of the pressure variations may vary fromabout 0.05 up to about 10 Hz, preferably 0.5 to 1 Hz. After the passagethrough the treating unit the biological material, e.g., whole blood,may reinserted in the patient directly, or may be stored for future use.Treated blood may be stored whole, or may be stored in its variouscomponents, e.g., plasma, platelets, erythrocytes, etc. Alternatively,the blood may be separated into its components prior to removal ofprions.

[0200] When the complexing agent is an antibody, it is often desirableto have a molecular spacer segment forming means for spacing theantibody from the wall of the exterior porous side of the hollow fibermembrane. This general arrangement is preferred when the molecularweight of the antigen is large, e.g., 100,000 Daltons or higher inmolecular weight. For example, a six or eight carbon methylene group isconvenient as a spacer or “handle” between antibody and membranesurface. When an antigen is readily absorbed by albumin or more readilychemically reacted with albumin than with the material of the filtermembrane surface, the spacer molecule may be a protein such a albumin.The outer surface of a membrane can be considered a relatively porousmaterial compared to that of the interior surface, which is normally theeffective filter surface of an ultrafilter membrane of the asymmetric,sometimes called anisotropic, type. Thus, for example, the exterior,porous side of a membrane may be treated with a 17% human albuminsolution in saline. The albumin will coat the surfaces within the porouszone of the membrane structure (i.e., the zone that underlies thebarrier layer of the membrane) and, thereafter, a solution of protein(e.g., a PrP^(Sc) antibody) can be deposited upon the albumin. Often itis desirable to cross-link the protein somewhat (as with a diluteglutaraldehyde solution or some other such mild cross-link-inducingagent); this aids in anchoring the material in place on the membranesurface.

[0201] One approach to preparing a cartridge that is capable of removingpathogenic factors from blood is an extracorporeal circulation systemwith fiber membranes having sufficient permeability for the pathogenicblood factor to be removed through the membrane and into a soluble,immobilized antibody sequestered in the extrafiber space. This involvesthe formation of a high molecular weight polymeric conjugate of thePrP^(Sc) antibody and PrP^(Sc) that cannot cross the filtration side ofthe membrane into the remainder of the biological sample, i.e., wherethe cells are maintained.

[0202] In order to form a soluble, immobilized complexing agent, themolecular weight of the immunoreactive complexing agent may be increasedto such a size that it will not diffuse, from the exterior, porousportion of the fiber and into the blood to be purified. This can be doneby chemically reacting the complexing agent with a high molecularweight, water-soluble substance such as silica gel or dextran or bypolymerizing the immunoreactive complexing agent. The use of suchmacromolecular-borne antibodies is advantageous for high rate of antigenabsorption, due to enhanced rate of polarization effects on masstransfer and binding kinetics.

[0203] Alternatively, the membrane may be composed of two membranehalves, which are mechanically generally identical to each other butwhich chemically may be built up of different material. In this case, itis enough if only the membrane half that faces away from the biologicalmaterial is able to bind to the prion complexing agent. For example, themembrane halves may be provided in an abutting relationship to eachother, wherein the PrP^(Sc) complexing agent preferably is bound in thepores and on both surfaces of the membrane half that faces away from thebiological material.

[0204] The complexing agent (e.g., NaPTA or anti-PrP^(Sc) antibodies)can also be immobilized in the membrane so that the surface that facestowards the biological material is free of the contacting reagent. Thisis to avoid contact between blood corpuscles and the reagent and therebypyrogen and/or anaphylactic reactions. Thus it is a form of a symmetricimmobilization, where on one surface of the membrane (as well as in thepores) the prion complexing agent is immobilized. The advantage ofimmobilizing within the pores of the membrane is that the activemicroscopic surface may be manifolded (>1000) compared to themacroscopic surface. Since the complexing agent is immobilized in thepart of membrane that faces away from the biological material thebiological material will not come into contact with the material.Consequently, any separate filtering of the biological material is notnecessary.

[0205] Alternatively, the prion complexing agent may be bound to aninsoluble matrix behind the membrane. The treating process is yetsimilar, but since the necessary diffusion distance is about 10 timeslonger, it may be necessary to arrange a somewhat more real flow throughthe membrane.

[0206] Irrespective of whether the prion complexing agent is immobilizedin the pores or immobilized to an insoluble matrix behind the membrane,the immobilizing procedure is preferably performed such that the complexof prions and the complexing agent remains bound and immobilized, i.e.,it is not present in the blood following the purification technique.Generally, covalent coupling is the safest immobilization. The nature ofcovalent coupling used depends on the choice of membrane material andthe nature of the complexing agent.

[0207] Magnetic Particles

[0208] Prions can also be removed from biological materials usingmagnetic particles comprised of prion complexing agent. The principlecomponents of the magnetic particles of the present invention are amagnetic core. The core consists of particles of iron oxide or othermagnetic materials. The PrP^(Sc) binding agent of the invention can beincorporated directly on the magnetic core, or indirectly incorporatedonto the magnetic core, e.g., through the use of a fibrous material anda binding agent. The fibrous material may comprise an organic polymer inthe form of fibers, such as carbohydrate polymers, urea formaldehyde orpolynonamethylene urea, and, in particular, cellulose fibers. Thebinding agent is a material which is introduced between the magneticcore and the fiber strands as a liquid, or in solution, and issolidified during the production process of freezing, polymerization orevaporation of a solvent. Examples of suitable binding agents are agar,gelatin, epoxy resin or urea formaldehyde furfuryl alcohol.

[0209] Magnetic microparticles useful in the present method can be avariety of shapes, which can be regular or irregular; preferably theshape maximizes the surface areas of the microparticles. The magneticmicroparticles should be of such a size that its separation fromsolution, for example by filtration or magnetic separation, is notdifficult. In addition, the magnetic microparticles should not be solarge that surface area is minimized or that they are not suitable formicroscale operations. Suitable sizes range from about 0.1.mu. meandiameter to about 100.mu. mean diameter. A preferred size is about1.0.mu. mean diameter. Suitable magnetic microparticles are commerciallyavailable from PerSeptive Diagnostics and are referred to as BioMag COOH(Catalog Number 8-4125).

[0210] The coated magnetic particles of the present invention can beproduced by stirring or mixing the core particles in a suspensioncomprising a fibrous material, a prion complexing agent, and a bindingagent. The fibers attach to the core particles and the binding agentfills the interstices. The binding agent is then solidified by one ofthe means as discussed above, in such a manner that the prion complexingagent is accessible on the outer surface. An example of such a system,which uses iron oxide as the core particles, cellulose fibers as thefibrous material and agar as the binding agent is described in U.S. Pat.No. 5,705,628.

[0211] The present invention also includes within its scope a compositemagnetic resin that comprises magnetic particles embedded in a organicpolymer matrix, which either contains, or has attached thereto, siteswhich are selective for prions.

[0212] The composite may thus comprise magnetic particles embedded in apolymeric resin which contains active sites or chemicals intended toselectively absorb to prions. For example, the polymeric resin has smallparticles of selective absorbers bound thereto. The selective absorbersmay be, for example, a metal salt of phosphotungtic acid.

[0213] The composite magnetic particles of the present invention may beused in a method for the removal of prions from any flowable biologicalsample. Removal of prions from the human product is by contacting thesolution to be treated with particles of a composite magnetic resin withimmobilized complexing agent and separating by magnetic filtration thecomposite magnetic resin particles from the solution. These magneticparticles may be used once and discarded, or recycled for use inpurifying other blood products. Particles can be recycled by subjectingthe separated composite magnetic resin particles to regeneration usingan appropriate regenerant solution, separating the regenerated compositemagnetic resin particles from the regenerant solution.

[0214] The composite magnetic resin particles with bound prions are thenselectively removed from the solution by magnetic filtration usingtechniques known in the art. The composite magnetic resin particles arethen recovered from the filter and the prions removed therefrom using anappropriate regenerant solution, for example an acidic solution. Thecleaned composite magnetic resin particles can then be recovered fromthe regenerant solution by magnetic filtration and the clean particlesrecycled for additional use.

[0215] Purification of biological material from a patient may be throughan extracorporal treatment unit, and following treatment the purifiedfluid may either be stored or may be reintroduced to the patient. Thebiological material is pumped from, for example, a patient into atreating unit comprising a microporous semi-permeable membrane havingpores of 0.01-0.8 microns, preferably 0.15-0.45 microns. During thepassage through the treating unit the biological material is exposed topressure variations (for example from −200 to +200 mmHg, preferably from−100 to +100 mmHg), whereby a penetrating fraction of the biologicalmaterial, e.g., the plasma, is caused to flow in an alternating paththrough the membrane wall in each direction for contacting thecomplexing agent.

[0216] Organs and Tissues for Transplantation

[0217] Organ, tissue and corneal transplantation are routine surgeriesin the United States. As with the blood supply, demand is far ahead ofsupply. More than 57,000 people are waiting for organ transplants. Manypeople who need transplants cannot get them because of the shortages;and some of them will die while waiting for a heart, liver or kidney. Anestimated 15,000 brain deaths occur in the United States each year, butonly about 30 percent donate their organs. It is possible to transplant25 different kinds of organs and tissues, including corneas, heart,heart valves, liver, kidneys, bone and cartilage, bone marrow, skin,pancreas, lungs, intestine, etc. In 1996, 4,058 livers were transplantedin the United States, compared with 164 in 1983. At least 9,000Americans, however, are still waiting for liver transplants. There were44,000 corneal transplants performed in the United States last year. Thenumber of Americans on waiting lists for corneas averages 5,000 at anygiven time. There were 6,500 skin grafts performed in 1996-1997.Transplanted skin is used an estimated 100,000 times per year asreplacement tissue for severe burns. Another 500,000 patients could havetheir wound-healing time shortened if enough skin were available.700,000 bone grafts were done in 1996. Transplanted bone tissue canreplace bone destroyed by tumors, trauma, and infection, healing limbsthat would otherwise have been amputated. Due to advances in medicaltechnology and improved preservation techniques, vital organs may betransported thousands of miles to a recipient. At the present time, anorgan donor in Great Britain introduces a potential risk of transmittingCJD to a transplant recipient.

[0218] Use of the present invention to treat organs and tissues prior totransplantation would increase the available pool of potential donatedorgans and tissues available for those in need, and would also denatureprions in any existing organs prior to transplantation.

[0219] A further aspect of the invention is to treat collagen before itis injected into a patient. Injectable collagen is a naturally producedprotein, derived from purified bovine collagen. It is used mostfrequently in cosmetic surgery, to minimize the appearance of wrinklesand scars, and to enlarge the lips. Injectable collagen is also used totreat stress incontinence in women by injecting it on either side of theurethra. For all of these procedures, the collagen is injected with afine-gauge needle under the skin where it is incorporated into thebody's own network of collagen fibers. The injections have to berepeated every 3-6 months to maintain the effect. Use of the claimedcompositions and methods to treat the collagen before it is injectedinto a patient would remove any possibility of transferring prions froman infected cow to a human.

[0220] The instant invention is not limited to human tissues and organsfor transplantation. Xenografts are also encompassed by the currentinvention. Suitable organs for transplant into humans may be found inbaboons, chimpanzees, pigs, sheep, etc. Although xenografts are stillexperimental, pig heart valves have been used for years as replacementsfor human heart valves. Clinical studies are ongoing with pig neuralcells being transplanted into Parkinson's disease patients. Thecontinuing progress in this area shows that the present invention may besuitable for application to animal tissues and organs before transplantinto a human recipient.

[0221] In summary, the invention comprises a composition that inhibitsprion formation or denatures existing prions in blood, blood products,organs and tissues. Blood, blood products, tissue or organs may bepre-treated to remove prions to the extent possible. Thereafter, acomposition of the invention is added to render any remaining prionsnon-infectious. The active component is added in an amount sufficient todenature any prions present in the blood, blood product, tissue or organto be transplanted. The amount will vary based on factors such as thetype of organ/tissue and its size. Generally, the organ or tissue willbe treated in a low pH (less than 5.0) solution, where the activeingredient is present in an amount about 0.1 to 1.0 μg per ml or mg ofmaterial to be treated. The treatment is for 2 hours or less, at atemperature of between 4° C. and 37° C.

[0222] ScN2a Cell-Based Assay

[0223] Another aspect of the invention is to assay compounds todetermine whether the compounds have the ability to denature infectiousproteins. On reading this disclosure, and in particular the descriptionprovided herein for particular assays, other assays will be apparent tothose skilled in the art. The basic concept is: (1) providing an acidcomponent in an aqueous and/or alcohol carrier, (2) adding the component(not known to be active), and (3) contacting the test composition with asample known to contain an infectious protein. After allowing time topass (e.g., 5 minutes to 2 hours) procedures described here are used to(4) determine if the component is “active” i.e., renders the proteinnon-infectious. Multiple compounds can be added to the composition andtested simultaneously. If no effect is found, then all of the compoundsare not active. If a sterilizing effect is found, the group of compoundstested can be divided in two and retested until an active compound isspecifically identified. Dividing the originally tested group in anymanner and retesting can be carried out any number of times.

[0224] The active component can be checked against one prion strain at atime or against multiple strains simultaneously. Some active componentssuch as SDS will inactivate all known strains of prion, while somepolycationic dendrimers will inactivate only specific strains. When theactive component inactivates all strains it is useful as an antisepticand/or therapeutic. When it inactivates only a specific strain it can beused to determine the strain of infectious prion in a sample.

[0225] Efforts were made to optimize the transfection of ScN2a cellswith pSPOX expression plasmids (Scott, M. R. et al., Protein Sci.1:986-997 (1992)). In connection with those effects, an evaluation wasmade of a transfection protocol that used SuperFect™ reagent (QIAGEN®).It was found that epitope-tagged (MHM2) PrP^(Sc) (Scott, M. R. et al.,Protein Sci. 1:986-997 (1992)) could not be detected in ScN2a cellsfollowing SuperFect-mediated transfection, whereas MHM2 PrP^(Sc) wasefficiently formed when a cationic liposome method or DNA delivery wasused. Close scrutiny revealed that, prior to protease digestion,SuperFect-transfected samples expressed MHM2 bands, which are not seenin the background pattern of an untransfected sample. The 3F4 monoclonalantibody does not react with MoPrP but does exhibit high backgroundstaining on Western blots of mouse ScN2a cells. Increased immunostainingin the 20-30 kDa region was observed compared to the non-transfectedsample. These observations led us to conclude that MHM2 PrP wassuccessfully expressed using SuperFect™ transfection reagent, but thatconversion of MHM2 PrP^(C) to protease-resistant MHM2 PrP^(Sc) wasinhibited by SuperFect™.

[0226] To investigate this apparent inhibition, a Western blot wasreprobed with RO73 polyclonal antiserum to detect endogenous MoPrP^(Sc),the presence of which is diagnostic for prion infection in ScN2a cells(Butler, D. A. et al., J. Virol. 62:1558-1564 (1988)). Surprisingly, itwas found that the SuperFect-treated ScN2a cells no longer containeddetectable quantities of MoPrP^(Sc). This result was also confirmed inWestern blots. To investigate the mechanism by which SuperFect™ reducedthe level of pre-existing PrP^(Sc) in chronically infected ScN2a cells,measurements were made of endogenous PrP^(Sc) in ScN2a cells exposed tovarious concentrations of SuperFect™ in the absence of plasmid DNA. Theresults showed that treatment with SuperFect™ (a branched polycation)caused the disappearance of PrP^(Sc) from ScN2a cells in adose-dependent manner. The concentration of SuperFect™ required toeliminate >95% of pre-existing PrP^(Sc) with a three hour exposure wasfound to be about 150 μg/ml. Duration of treatment also influenced theability of SuperFect™ to remove PrP^(Sc) from ScN2a cells: exposure to150 μg/ml SuperFect™ for 10 min did not affect PrP^(Sc) levels, whereas7.5 μg/ml SuperFect™ eliminated all detectable PrP^(Sc) with a t1/2=8 h.

[0227] SuperFect™ is a mixture of branched polyamines derived fromheat-induced degradation of a PAMAM dendrimer (Tang, M. X. et al.,Bioconjug. Chem. 7:703-714 (1996)). Knowing this structure the abilityof several other branched and unbranched polymers to eliminate PrP^(Sc)from ScN2a cells were tested (see Table 2, below). The branched polymersinvestigated include various preparations of PEI, as well as intactPAMAM and PPI dendrimers. Dendrimers are manufactured by a repetitivedivergent growth technique, allowing the synthesis of successive,well-defined “generations” of homodisperse structures (FIG. 1). Thepotency of both PAMAM and PPI dendrimers in eliminating PrP^(Sc) fromScN2a cells increased as the generation level increased. The most potentcompounds with respect to eliminating PrP^(Sc) were PAMAM generation 4.0and PPI generation 4.0, whereas PAMAM generation 1.0 showed very littleability to eliminate PrP^(Sc) (see Table 2). Similarly, a high MWfraction of PEI was more potent than low MW PEI.

[0228] From the foregoing data, it is clear that for all three branchedpolyamines tested, increasing molecular size corresponded to anincreased potency for eliminating PrP^(Sc). To determine whether thistrend was directly attributable to increased surface density of aminogroups on the larger molecules, PAMAM-OH generation 4.0 was tested. Thisis a dendrimer that resembles PAMAM generation 4.0 except that hydroxylsreplace amino groups on its surface. Unlike PAMAM generation 4.0,PAMAM-OH generation 4.0 did not cause a reduction of PrP^(Sc) levelseven at the highest concentration tested (10 mg/ml), establishing thatthe amino groups are required for the elimination of PrP^(Sc) by PAMAM(Table 2).

[0229] In an effort to assess the contribution of the branchedarchitecture to the clearing ability of polyamines for PrP^(Sc), thelinear molecules poly-(L)lysine and linear PEI were also tested. Both ofthese linear compounds were less potent than a preparation of branchedPEI with similar average molecular weight (Table 2), establishing that abranched molecular architecture optimizes the ability of polyamines toeliminate PrP^(Sc), presumably because the branched structures achieve ahigher density of surface amino groups.

[0230] Kinetics of PrP^(Sc) Elimination by Polyamines

[0231] The preceding results demonstrate the potent ability of branchedpolyamines to clear PrP^(Sc) from ScN2a cells within a few hours oftreatment. The utility of these compounds to act as therapeutics fortreatment of prion disease was tested by determining whether they werecytotoxic for ScN2a cells, using as criteria cell growth, morphology,and viability as measured by trypan blue staining. None of the compoundswas cytotoxic to ScN2a cells after exposure for one week atconcentrations up to 7.5 μg/ml. To determine whether branched polyaminescan cure ScN2a cells of scrapie infection without affecting cellviability, the kinetics of prion clearance was examined in the presenceof a non-cytotoxic concentration (7.5 μg/ml) of three different branchedpolyamines. ScN2a cells were exposed to SuperFect™, PEI, or PAMAMgeneration 4.0 for varying periods of time. The kinetics of PrP^(Sc)elimination was assessed by Western blotting. All three compounds causeda substantial reduction in PrP^(Sc) levels after 8-16 h of treatment,and of the three compounds, PEI appeared to remove PrP^(Sc) mostquickly, with a t1/2=4 h.

[0232] Curing Neuroblastoma Cells of Scrapie Infection

[0233] The above results show that it is possible to reverse theaccumulation of PrP^(Sc) in ScN2a cells under non-cytotoxic conditions.It was also found that extended exposure to even lower levels of thebranched polyamines (1.5 μg/ml) was sufficient to eliminate PrP^(Sc).Based on these findings, this protocol was used to determine whether thesevere reduction in PrP^(Sc) levels following exposure to branchedpolyamines would persist after removal of the compounds. Following theexposure of ScN2a cells to a 1.5 μg/ml SuperFect™ for 1 week, PrP^(Sc)was reduced to <1% of the baseline level, but then increased back to ˜5%of the baseline level after 3 additional weeks in culture in the absenceof polyamine. In contrast, following exposure to 1.5 μg/ml of either PEIor PAMAM generation 4.0 for 1 week, PrP^(Sc) was completely eliminatedand did not return even after 3 weeks in culture without polyamines. Amore intensive course of treatment with 1.8 μg/ml SuperFect™ for 9 dalso cured ScN2a cells of scrapie infection fully, manifested by theabsence of PrP^(Sc) 1 month after removal of SuperFect™.

[0234] Evidence For Polyamines Acting Within An Acidic Compartment

[0235] The above results showed the potent activity of branchedpolyamines in rapidly clearing scrapie prions from cultured ScN2a cells.Based on these results, the mechanism by which these compounds act wasinvestigated. All of the compounds which effect removal of PrP^(Sc) fromScN2a cells are known to traffic through endosomes (Boussif, O. et al.,Proc. Natl. Acad. Sci. USA 92:7297-7301 (1995); and Haensler, J. &Szoka, F. C. J., Bioconjug. Chem. 4:372-379 (1993)). Since PrP^(C) isconverted into PrP^(Sc) in caveolae-like domains (CLDs) or rafts(Gorodinsky, A. & Harris, D. A., J. Cell Biol. 129:619-627 (1995);Taraboulos, A. et al., J. Cell Biol. 129:121-132 (1995); Vey, M. et al.,Proc. Natl. Acad. Sci. USA 93:14945-14949 (1996); and Kaneko, K. et al.,Proc. Natl. Acad. Sci. USA 94:2333-2338 (1997)) and is then internalizedthrough the endocytic pathway (Caughey, B. et al., J. Virol.65:6597-6603 (1991); and Borchelt, D. R. et al., J. Biol. Chem.267:16188-16199 (1992)), it was deduced that polyamines act uponPrP^(Sc) in endosomes or lysosomes. This deduction was investigated bydetermining the effect of pretreatment with the lysosomotropic agentschloroquine and NH₄Cl on the ability of polyamines to eliminatePrP^(Sc). These lysosomotropic agents alkalinize endosomes and have noeffect on PrP^(Sc) levels when administered to ScN2a cells (Taraboulos,A. et al., Mol. Biol. Cell 3:851-863 (1992)). Experimental resultsobtained show that 100 μM chloroquine, but not 30 μM NH₄Cl, blocked theability of PEI to eliminate PrP^(Sc). Similar results were obtained withSuperFect™ and PAMAM, generation 4.0. Although the failure of NH₄Cl toaffect PrP^(Sc) levels is not easily explained, the ability ofchloroquine to attenuate the ability of branched polyamines to removePrP^(Sc) is consistent with the notion that these agents act inendosomes or lysosomes.

[0236] Organ Homogenate Assay

[0237] The above results with cell cultures prompted investigating thepossibility that in an acidic environment branched polyamines, either byindirectly interacting with PrP^(Sc) or with another cellular component,could cause PrP^(Sc) to become susceptible to hydrolases present in theendosome/lysozome. An in vitro degradation assay was developed toevaluate the effect of pH on the ability of polyamines to renderPrP^(Sc) sensitive to protease. Crude homogenates of scrapie-infectedmouse brain were exposed to a broad range of pH values in the presenceor absence of SuperFect™ and then treated with proteinase K prior toWestern blotting. Whereas PrP^(Sc) remained resistant to proteasehydrolysis throughout the pH range (3.6-9.6) in the absence ofSuperFect™, addition of the branched polyamine at pH 4.0 or below causedPrP^(Sc) to become almost completely degraded by protease.

[0238] Polyamine addition showed a dramatic effect on clearance in vitrowhich was optimized at pH 4 or less. These results show that polyaminesact on PrP^(Sc) in an acidic compartment. To establish that the in vitrodegradation assay is a valid approximation of the mechanism by whichbranched polyamines enhance the clearance of PrP^(Sc) from culturedcells, a structure activity analysis was performed with several of thecompounds tested in culture cells. An excellent correlation was foundbetween the clearance of Prp^(Sc) in cultured ScN2a cells (Table 2) andthe ability to render PrP^(Sc) susceptible to protease at acidic pH invitro. Notably, PAMAM-OH generation 4.0 failed to render PrP^(Sc)susceptible to protease, whereas PAMAM generation 4.0 and PPI,generation 4.0 exhibited an even stronger activity than SuperFect™ invitro, as expected from their observed potency in cultured ScN2a cells(Table 2).

[0239] Mechanism of Action

[0240] When a pH of 4.0 or less was maintained the results show thatcertain branched polyamines cause the rapid elimination of PrP^(Sc) fromScN2a cells in a dose- and time-dependent manner. These compoundsdemonstrate a potent ability to remove prions from cultured-cells atconcentrations that are completely non-cytotoxic. The cells may bemaintained indefinitely in culture in the presence of therapeutic levelsof branched polyamines. Furthermore, when ScN2a cells were exposed tothese compounds for ˜1 week, PrP^(Sc) was reduced to undetectable levelsand remained so for at least one month after removal of the polyamine.

[0241] Clarification of the exact mechanism of PrP^(Sc) elimination bybranched polyamines is an important objective. Although a number ofpossible scenarios exist, several possibilities may be excluded already.One possibility that was eliminated was that polyamines act by inductionof chaperones such as heat shock proteins that mediate prion proteinrefolding because the above results show that it was possible toreproduce the phenomenon in vitro. Furthermore, polyamines seem to offeradvantages over other putative therapeutics that would seek to promoterefolding: at very high concentrations, dimethyl sulfoxide (DMSO) andglycerol act as direct “chemical chaperones” and inhibit the formationof new PrP^(Sc) (Tatzelt, J. et al., EMBO J. 15:6363-6373 (1996)), butthese compounds cannot reduce pre-existing PrP^(Sc) levels. Furthermore,polyamines inhibit PrP^(Sc) formation at much lower concentrations thanthese agents. The ability of polyamines to effect the rapid clearance ofPrP^(Sc) also contrasts with the activity of other potential priontherapeutics. Sulfated polyanions may inhibit PrP^(Sc) accumulation inScN2a cells by directly binding to PrP^(C) (Gabizon, R. et al., J. Cell.Physiol. 157 (1993); Caughey, B. et al., J. Virol. 68:2135-2141 (1994)),but because branched polyamines are able to clear pre-existing PrP^(Sc),their mechanism of action cannot simply involve binding to PrP^(C) andinhibiting de novo synthesis.

[0242] Another possible mechanism which can be excluded is endosomalrupture. The branched polyamines which were effective in clearingPrP^(Sc) from ScN2a cells in our experiments, PEI, SuperFect™ and PAMAM,are also potent lysosomotropic, osmotic agents which can swell in acidicenvironments and rupture endosomes (Boussif, O. et al., Proc. Natl.Acad. Sci. USA 92:7297-7301 (1995); Haensler, J. & Szoka, F. C. J.,Bioconjug. Chem. 4:372-379 (1993)). This might suggest that branchedpolyamines clear PrP^(Sc) from ScN2a cells by rupturing endosomes andexposing PrP^(Sc) to cytosolic degradation processes. However, it isknown that the lysosomotropic, endosome-rupturing agents NH₄Cl,chloroquine, and monensin do not interfere with the formation ofPrP^(Sc) in ScN2a cells (Taraboulos, A. et al., Mol. Biol. Cell3:851-863 (1992)). Furthermore, the results also show that chloroquineinterferes with the ability of branched polyamines to clear PrP^(Sc) andthat polyamines can clear PrP^(Sc) in vitro at acidic pH in the absenceof cell membranes. Together, these observations rule out endosomerupture as the mechanism by which branched polyamines remove PrP^(Sc).

[0243] Without committing to any particular mechanism of action itappears likely that branched polyamines require the acidic environmentof intact endosomes or lyzosomes to destroy PrP^(Sc). Thestructure-activity profile of polymers tested reveals that the mostactive compounds possess densely packed, regularly-spaced amino groups,suggesting that these compounds may bind to a ligand which hasperiodically-spaced negative charges. Several scenarios remain possible:(1) Branched polyamines may bind directly to PrP^(Sc) arranged as anamyloid with exposed negatively-charged moieties and induce aconformational change under acidic conditions; (2) Treatment of PrP27-30 with acid decreases turbidity and increases α-helical content,suggesting that such conditions might dissociate PrP^(Sc) into monomers(Safar, J., Roller, P. P., Gajdusek, D. C. & Gibbs, C. J., Jr. Scrapieamyloid (prion) protein has the conformational characteristics of anaggregated molten globule folding intermediate). It is thereforepossible that polyamines bind to an equilibrium unfolding intermediateof PrP^(Sc) present under acidic conditions. (3) Alternatively,polyamines might sequester a cryptic, negatively charged component boundto PrP^(Sc) that is essential for protease resistance, but which is onlyreleased when PrP^(Sc) undergoes an acid-induced conformational change.Such a component might act as a chaperone for PrP^(Sc) inside endosomesor lysosomes. (4) Finally, another possibility is that polyaminesactivate an endosomal or lysosomal factor which can induce aconformational change in Prp^(Sc). Clearly, more work will be requiredto determine the precise mechanism by which branched polyamines destroyPrP^(Sc).

[0244] General Applicability of Assay

[0245] The in vitro assay described here is generally applicable in thesearch for compounds that effectively clear conformationally alteredproteins present in food thereby preventing a number of degenerativediseases, where the accumulation of proteins seems to mediate thepathogenesis of these illnesses. By simulating lysosomes, whereproteases hydrolyze proteins under acidic conditions, the in vitro brainhomogenate assay is able to rapidly evaluate the efficacy of a varietyof polyamines to induce degradation of PrP^(Sc).

[0246] The in vitro assay which used scrapie infected brain homogenateto test for compounds which clear PrP^(Sc) could be modified to assayfor compounds which would clear any conformationally altered protein.The assay is carried out by homogenizing the organ or tissue where theconformationally altered protein is present in the highestconcentration. The pH of the homogenate is then reduced to less than 5.0and preferably 4.0 or less. For example, pancreatic tissue can behomogenized to produce an assay to test for compounds that clear amylin,which is associated with type II Diabetes. Homogenized kidney could beused to test for compounds which clear β₂-microglobulin and homogenizedheart or vascular tissue used to test for compounds which clear atrialnatriuretic factor. Those skilled in the art will recognize other organsand tissue types that can be homogenized to test for other compoundsthat clear other conformationally altered proteins.

[0247] Besides using the in vitro assay to screen for potential drugs,the compounds found via the assay such as branched polyamines provide anew tool for exploring the conversion of a protein to conformationallyaltered protein, e.g., PrP^(C) into PrP^(Sc). The mechanism by whichbranched polyamines render PrP^(Sc) susceptible to proteolysis, remainsto be established. Whether the interaction of branched polyamines withPrP^(Sc) is reversible is unknown. In addition, it is not known whetherbranched polyamines are able to solubilize PrP^(Sc) without irreversiblydenaturing the protein. Whatever the mechanism by which branchedpolyamines interact with PrP^(Sc), it is likely to be different fromthat found with chaotropes as well as denaturing detergents and solvents(Prusiner, S. B. et al., Proc. Natl. Acad. Sci. USA 90:2793-2797(1993)).

[0248] Using the assays described and disclosed here certain specificbranched polyamines have been found which mediate the clearance ofPrP^(Sc) from cultured cells under non-cytotoxic conditions. Thesecompounds offer the possibility of being added to a wide range of low pHfood products to neutralize conformational altered proteins present.Since the compounds act by stimulating normal cellular pathways ofprotein degradation to destroy PrP^(Sc), this class of compounds wouldalso likely be of value in the treatment of other degenerative andhereditary disorders where abnormally folded, wild-type or mutantproteins accumulate. Such an approach may find merit in developing aneffective therapeutics for one or more of the common, degenerativeillnesses including Alzheimer's disease, Parkinson's disease,amyotrophic lateral sclerosis, frontotemporal dementia, adult onsetdiabetes mellitus and the amyloidoses (Beyreuther, K. & Masters, C. L.,Nature Medicine 3:723-725 (1997); Masters, C. L. & Beyreuther, K., BMJ316:446-448 (1998); Selkoe, D. J., Trends in Cell Biol. 8:447-453(1998); Selkoe, D. J., Nature 399:A23-31 (1999); Wong, P. C. et al.,Neuron 14:1105-1116 (1995); Spillantini, M. G. et al., Proc. Natl. Acad.Sci. USA 95:6469-6473 (1998); Hutton, M., et al., Nature 393:702-705(1998); and Stone, M. J., Blood 75:531-545 (1990)). Whether branchedpolyamines might also prove efficacious in a variety of inheriteddisorders where the accumulation of abnormal proteins is a hallmark ofthe illness remains to be established; these genetic maladies includeheritable forms of prion disease, Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis, frontotemporal dementia, Pick'sdisease and amyloidosis, as well as the triplet repeat diseasesincluding Huntington's disease, spinal cerebellar ataxias and myotonicdystrophy (Fu, Y.-H. et al., Science 255:1256-1259 (1992); and Group,T., Cell 72:971-983 (1993)). Compounds identified via assays of theinvention such as branched polyamines will find utility in preventing ordelaying the onset of these genetic diseases where carriers can often beidentified decades in advance of detectable neurologic or systemicdysfunction.

[0249] The invention is based on the discovery that several dendriticpolycations, including the starburst dendrimers SuperFect™ (QIAGEN®),Valencia, Calif.), polyamidoamide (PAMAM), and the hyperbranchedpolycation polyethyleneimine (PEI), were surprisingly found to eliminatePrP^(Sc) from cultured scrapie-infected neuroblastoma cells. Thesehighly-branched, polycationic compounds provide a novel class oftherapeutic agents to combat prion diseases and other degenerativedisease including the amyloidoses. The removal of PrP^(Sc) is dependenton both the concentration of dendritic polymer and length of exposure.Dendritic polymers were able to clear PrP^(Sc) at concentrations whichwere not cytotoxic. Repeated exposures to heat-degraded starburst PAMAMdendrimer or PEI caused a dramatic reduction in PrP^(Sc) levels, whichpersisted for a month even after removal of the compound. Dendriticpolycations did not appear to destroy purified PrP^(Sc) in vitro, andtherefore may act through a generalized mechanism. Dendritic polycationsrepresent a class of compounds which can be used as therapeutic agentsin prion diseases and other disorders involving insoluble proteindeposits, such as the amyloidoses.

EXAMPLES

[0250] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the present invention, and are not intended to limitthe scope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

[0251] Methods And Materials

[0252] Chemicals. High molecular weight PEI was purchased from Fluka.DOTAP cationic lipid was purchased from Boehringer Mannheim andSuperFect™ transfection reagent was purchased from QIAGEN®. All othercompounds were purchased from Sigma-Aldrich. All test compounds weredissolved in water at stock concentration of 3 mg/ml and filteredthrough a Millipore 0.22 mm filter.

[0253] Cultured cells. Stock cultures of ScN2a cells were maintained inMEM with 10% FBS, 10% Glutamax (Gibco BRL), 100 U penicillin, and 100mg/ml streptomycin (supplemented DME). Immediately prior to addition oftest compounds, the dishes were washed twice with fresh supplemented DMEmedia. After exposure to test compounds, dishes were drained of mediaand cells were harvested by lysis in 0.25-1 ml 20 mM Tris pH 8.0containing 100 mM NaCl, 0.5% NP-40, and 0.5% sodium deoxycholate toobtain a total protein concentration of 1 mg/ml measured by the BCAassay. Nuclei were removed from the lysate by centrifugation at 2000 rpmfor 5 min. For samples not treated with proteinase K, 40 μl of wholelysate (representing 40 μg total protein) was mixed with an equal volumeof 2×SDS reducing sample buffer. For proteinase K digestion, 20 μg/mlproteinase K (Boehringer Mannheim) (total protein:enzyme ratio 50:1) wasadded, and the sample was incubated for 1 h at 37° C. Proteolyticdigestion was terminated by the addition of Pefabloc to a finalconcentration of 5 mM. One ml samples were centrifuged at 100,000×g for1 h at 4° C., the supernatants were discarded, and the pellets wereresuspended in 80 μl of reducing SDS sample buffer for SDS-PAGE.

[0254] Brain homogenates. Brain homogenates from RML scrapie-affectedCD-1 mice (10% (w/v) in sterile water) were prepared by repeatedextrusion through syringe needles of successively smaller size, from 18to 22 gauge. Nuclei and debris were removed by centrifugation at 1000×gfor 5 min. The bicinchnoninic acid (BCA) protein assay (Pierce) was usedto determine protein concentration. Homogenates were adjusted to 1 mg/mlprotein in 1% NP-40. For reactions, 0.5 ml homogenate was incubated with25 ml 1.0 M buffer (sodium acetate for pH 3-6 and Tris acetate for pH7-10) plus or minus 10 ml of polyamine stock solution (3 mg/ml) for 2 hat 37° C. with constant shaking. The final pH value of each sample wasmeasured directly with a calibrated pH electrode (RadiometerCopenhagen). Following incubation, each sample was neutralized with anequal volume 0.2 M HEPES pH 7.5 containing 0.3 M NaCl and 4% Sarkosyl.Proteinase K was added to achieve a final concentration of 20 μg/ml, andsamples were incubated for 1 h at 37° C. Proteolytic digestion wasterminated by the addition of Pefabloc to a final concentration of 5 μM.Ten μl of digested brain homogenate was mixed with equal volume 2×SDSsample buffer and analyzed by SDS-PAGE followed by Western blotting.

[0255] Western blotting. Following electrophoresis, Western blotting wasperformed as previously described (Scott, M. et al., Cell 59:847-857(1989)). Samples were boiled for 5 min and cleared by centrifugation for1 min at 14,000 rpm in a Beckman ultrafuge. SDS-PAGE was carried out in1.5 mm, 12% polyacrylamide gels (Laemmli, U.K., Nature 227:680-685(1970)). Membranes were blocked with 5% non-fat milk protein in PBST(calcium- and magnesium-free PBS plus 0.1% Tween 20) for 1 h at roomtemperature. Blocked membranes were incubated with primary RO73polyclonal antibody (to detect MoPrP) (Serban, D. et al., Neurology40:110-117 (1990)) or 3F4 monoclonal antibody (to detect MHM2 PrP)(Kascsak, R. J. et al., J. Virol. 61:3688-3693 (1987)) at 1:5000dilution in PBST overnight at 4° C. Following incubation with primaryantibody, membranes were washed 3×10 min in PBST, incubated withhorseradish peroxidase-labeled secondary antibody (Amersham LifeSciences) diluted 1:5000 in PBST for 30 to. 60 min at 4° C. and washedagain for 3×10 min in PBST. After chemiluminescent development with ECLreagent (Amersham) for 1 min, blots were sealed in plastic covers andexposed to ECL Hypermax film (Amersham). Films were processedautomatically in a Konica film processor.

Example 1A Branched Polyamines Inhibit Formation of Nascent PrP^(Sc) andInduce Clearance of Pre-Existing PrP^(Sc)

[0256] Western blots were probed with 3F4 monoclonal antibody whichrecognizes newly expressed MHM2 PrP. ScN2a cells were exposed toSuperFect™ for 3 h and harvested 3 d after removal of SuperFect™. Gelswere run on both undigested, control sample and a sample subjected tolimited proteolysis. The samples were run in separate lanes 1-6 with acontrol and limited proteolysis sample for each of the 6 lanes asfollows: Lane 1: DOTAP-mediated transfection. Lane 2: 30 μg/mlSuperFect™, 5 μg pSPOX MHM2. Lane 3: 75 μg/ml SuperFect™, 5 μg pSPOXMHM2. Lane 4:150 μg/ml SuperFect™, 5 μg pSOX MHM2. Lane 5: 150 μg/mlSuperFect™, 10 μg pSPOX MHM2. Lane 6: No addition of either transfectionreagent or DNA. Forty μl of undigested brain homogenate was used inthese studies while those samples subjected to limited digestion withproteinase K were concentrated 25-fold prior to SDS-PAGE. One ml of thedigest were centrifuged at 100,000×g for 1 h at 4° C. and the pelletssuspended in 80 μl of SDS sample buffer prior to SDS-PAGE followed byWestern blotting. Apparent molecular weights based on migration ofprotein standards are 34.2, 28.3, and 19.9 kDa.

[0257] All of the control lanes 1-6 show multiple bands as expected.However, of the samples subjected to limited proteolytic only lane 1shows bands. Unexpectedly, all of the partially digested sample lanes2-5 show no bands and as expected no bands in the partially digestedlane 6. These results show the effect of using SuperFect™ in clearingPrP^(Sc).

Example 1B

[0258] The blot described above was stripped of antibody, exposed tolabeled RO73 and redeveloped. The antibody 3F4 used in Example 1 bindsto PrP^(C) but not to PrP^(Sc). However, RO73 binds to PrP^(Sc) andPrP^(C). Lanes 1, 2 and 3 show decreasing amounts of PrP and lanes 4 and5 show no detectable PrP^(Sc).

Example 2A

[0259] Gels were run on undigested controls 1-4 and as above, samplessubjected to limited proteolysis. The lanes were as follows: Lane 1: NoSuperFect™. Lane 2: 30 μg/ml SuperFect™. Lane 3: 75 μg/ml SuperFect.Lane 4: 150 μg/ml SuperFect. ScN2a cells were exposed to SuperFect™ for3 h and harvested 3 d after removal of SuperFect™. Apparent molecularweights based on migration of protein standards are 33.9, 28.8, and 20.5kDa. In that each sample was tested after the same time period theresults show the dose-dependent effect of SuperFect™ on PrP^(Sc)removal. Lanes 1, 2 and 3 show decreasing amounts of PrP^(Sc) and lane 4shows no detectable PrP^(Sc).

Example 2B

[0260] To determine the time-dependent effect of SuperFect™ threedifferent panels with four lanes each were prepared and run as follows:ScN2a cells were exposed to 7.5 μg/ml: SuperFect™ (lanes 1-4), PEI(average molecular weight ˜60,000) (lanes 5-8), or PAMAM, generation4.0. (lanes 9-12). Time of exposure times for each polyamine: 0 hours(lanes 1, 5, and 9), 4 hours (lanes 2, 6, and 10), 8 hours (lanes 3, 7,and 11), 16 hours (lanes 4, 8, and 12). All samples were subjected tolimited proteolysis to measure PrP^(Sc). Apparent molecular weightsbased on migration of protein standards are 38, 26, and 15 kDa. Lanes ofeach of the three panels show decreasing amounts of PrP^(Sc).

Example 3

[0261] In this example four panels A, B, C and D were created withpanels having three double (control and test) lanes each. ScN2a cellswere exposed to 1.5 μg/ml: (A) SuperFect™, (B) PEI (average molecularweight ˜60,000), (C) PAMAM, generation 4.0, or (D) no addition. Cellswere harvested: Lane 1 , before addition; Lane 2 , immediately following1 week continuous exposure to test compounds; and Lane 3 , three weeksafter removal of test compounds. Minus (−) symbol denotes undigested,control sample and plus (+) symbol designates sample subjected tolimited proteolysis. Apparent molecular weights based on migration ofprotein standards are 33.9, 28.8, and 20.5 kDa. Test lanes 3 in panel Ashowed slight PrP^(Sc) after three weeks and test lanes 3 in panels Band C showed no detectable PrP^(Sc) whereas PrP^(Sc) was present in alllanes in panel D.

Example 4A

[0262] Four separate gels were run to demonstrate the effect of addingchloroquine would have on PrP^(Sc) levels. The lanes 1 control and 3where chloroquine was added show clear bands for PrP^(Sc) whereas lanes2 and 4 with no chloroquine show barely detectable amounts of PrP^(Sc).The four lanes were prepared as follows: ScN2a cells were treated Lane 1: Control media. Lane 2: 7.5 μg/ml PEI (average molecular weight˜60,000). Lane 3: PEI plus 100 μM chloroquine. Lane 4: PEI plus 30 μMNH₄Cl. Chloroquine and NH₄Cl were added 1 h prior to addition of PEI.Cells were harvested 16 hours after addition of PEI. All samples shownwere subjected to limited proteolysis to measure PrP^(Sc). Apparentmolecular weights based on migration of protein standards are 38, 26,and 15 kDa.

Example 4B

[0263] Eight lanes with SuperFect™ (+SF) and eight lanes withoutSuperFect™ (−SF) were prepared. Lanes 1-8 of each group had an adjustedpH of 3.6, 4, 5, 6, 7, 8, 9 and 9.6. In vitro mixture of crude mousebrain homogenates with SuperFect™ under a range of pH conditions wasperformed as described in methods (measured final pH of each sampledenoted above the lanes). Addition of 60 μg/ml SuperFect™ denoted as“+SF” and control with no addition as “−SF.” All samples shown weresubjected to limited proteolysis to measure PrP^(Sc). Apparent molecularweights based on migration of protein standards are 30 and 27 kDa. Alllanes of the −SF group showed PrP^(Sc) present. Lanes 3-8 of the +SFgroup showed PrP^(Sc). However, lanes 1 and 2 with respective pH levelsof 3.6 and 4.0 showed very slight detectable PrP^(Sc). The results showthat the ability of a blanched polycation such as SuperFect™ to clearPrP^(Sc) is pH dependent.

Example 5

[0264] Sixteen different lanes were prepared as described. Lanes 1 and 2were control lanes and each of lanes 3-16 contained a different compoundas tested in Table 2. The test compounds were all polyamines. Thus, theresults show removal of PrP^(Sc) from brain homogenate in vitro byvarious polyamines. Samples were incubated with polyamines at pH 3.6 andprocessed as described in Methods. Each polyamine was tested at 60 μg/mlconcentration. Lanes 1 and 2: control. Lane 3: poly-(L)lysine. Lane 4:PAMAM, generation 0.0. Lane 5: PAMAM, generation 1.0. Lane 6: PAMAM,generation 2.0. Lane 7: PAMAM, generation 3.0. Lane 8: PAMAM, generation4.0. Lane 9: PAMAM-OH, generation 4.0. Lane 10: PPI, generation 2.0.Lane 11: PPI, generation 4.0. Lane 12: linear PEI. Lane 13: high MW PEI.Lane 14: low MW PEI. Lane 15: average MW PEI. Lane 16: SuperFect. Allsamples shown were subjected to limited proteolysis to measure PrP^(Sc).Apparent molecular weights based on migration of protein standards are30 and 27 kDa. Table 2. Removal of PrP^(Sc) by polymer compounds.IC₅₀=approximate concentration of polymer required to reduce PrP^(Sc) to50% of control levels in ScN2a cells after exposure for 16 hours. Allcompounds were tested at 5 different concentrations. PrP^(Sc) levelswere measured by densitometry of Western blot signals. TABLE 2 (includesinformation on the characteristics of compounds used but does notcorrespond directly to lanes 1-16) Compound Mol. Wt. Primary NH₂ groupsIC₅₀ (ng/ml) PAMAM generation 0.0 517 4 >10,000 PAMAM generation 1.01,430 8 >10,000 PAMAM generation 2.0 3,526 16 2,000 PAMAM generation 3.06,909 32 400 PAMAM generation 4.0 14,215 64 80 PAMAM-OH generation14,279 0 >10,000 PPI generation 2.0 773 8 2,000 PPI generation 4.0 3,51432 80 Low MW PEI ˜25,000 2,000 Average MW PEI ˜60,000 400 High MW PEI˜800,000 80 Linear PEI ˜60,000 2,000 poly-(L)lysine ˜60,000 >500 10,000SuperFect ™ 400

[0265] Lanes 7, 8, 11 and 13 showed the best results, i.e., best abilityto clear PrP^(Sc) under these conditions. Specifically, PAMAM generation4.0 in lane 8 showed the best ability to clear PrP^(Sc) under theseconditions whereas PAMAM-OH generation 4.0 showed almost no detectableability to clear PrP^(Sc) and was comparable to the control.

Example 6 Transfection of PrP^(Sc) Expressing Cells With DendrimerCompounds

[0266] Cells of neuronal origin expressing PrP^(Sc) were examined forthe ability of compounds to suppress PrP^(Sc) formation. Stock culturesof N2a and ScN2a cells were maintained in MEM with 10% FBS, 10% Glutamax(Gibco BRL), 100 U penicillin, and 100 μg/ml streptomycin. Cells from asingle confluent 100 mm dish were trypsinized and split into 10 separate60 mm dishes containing DME plus 10% FBS, 10% Glutamax, 100 Upenicillin, and 100 μg/ml streptomycin (supplemented DME) one day priorto transfection. Immediately prior to transfection, the dishes werewashed twice with 4 ml supplemented DME media and then drained.

[0267] For DOTAP-mediated transfection, 15 g pSPOX MHM2 was resuspendedin 150 μl sterile Hepes Buffered Saline (HBS) on the day oftransfection. The DNA solution was then mixed with an equal volume of333 μg/ml DOTAP (Boehringer Mannheim) in HBS in Falcon 2059 tubes andincubated at room temperature for 10 minutes to allow formation ofDNA/lipid complexes. Supplemented DME (2.5 ml) was added to the mixture,and this was then pipetted onto drained cell monolayers. The followingday, the medium containing DNA/lipid was removed and replaced with freshsupplemented DME. Cells were harvested three days later.

[0268] For SuperFect™-mediated transfections/exposures, SuperFect™ withor without DNA was added to 1 ml supplemented DME in a Falcon 2059 tubeto achieve the specific concentrations needed for each experiment. Thismixture was pipetted up and down twice and then onto drained cellmonolayers. After exposure for the indicated times, the mediumcontaining SuperFect™ was removed and replaced with fresh supplementedDME. Cells were harvested at specified times after removal ofSuperFect™.

[0269] Exposures to PPI (DAB-Am-8, Polypropylenimine octaamineDendrimer, Generation 2.0 Aldrich 46,072-9), Intact PAMAM (Starburst(PAMAM) Dendrimer, Generation 4.

[0270] Aldrich 41,244-9, PEI (Sigma), poly-(L)lysine (Sigma), andpoly-(D) lysine (Sigma) were performed as described above forSuperFect™.

[0271] Isolation of Protein from Treated Cells

[0272] Cells were harvested by lysis in 1.2 ml of 20 mM Tris pH 8.0containing 100 mM NaCl, 0.5% NP-40, add 0.5% sodium deoxycholate. Nucleiwere removed from the lysate by centrifugation at 2000 rpm for 5 min.This lysate typically had a protein concentration of 0.5 mg/ml measuredby the BCA assay. For samples not treated with proteinase K, 40 μl ofwhole lysate (representing 20 μg total protein) was mixed with 40 μl of2×SDS sample buffer. For proteinase K digestion, 1 ml of lysate wasincubated with 20 μg/ml proteinase K (total protein:enzyme ratio 25:1)for 1 hr at 37° C. Proteolytic digestion was terminated by the additionof 8 μl of 0.5M PMSF in absolute ethanol. Samples were then centrifugedfor 75 min in a Beckman TLA-45 rotor at 100,000×g at 4° C. The pelletwas resuspended by repeated pipetting in 80 μl of 1×SDS sample buffer.The entire sample (representing 0.5 mg total protein before digestion)was loaded for SDS-PAGE.

[0273] Western Blot Analysis

[0274] Immunoreactive PrP bands from the DOTAP-mediated transfectionwere detected before and after digestion with proteinase K withmonoclonal antibody 3F4. The construct used to express PrP^(Sc) in theScN2a cells is MHM2 a chimeric construct that differs from wild-type(wt) MoPrP at positions 108 and 11 (Scott et al. Protein Sci. 1:986-997(1992)). Substitution at these positions with the corresponding residues(109 and 112 respectively) from the Syrian hamster (SHa) PrP sequencecreates an epitope for 3F4 (Kascsak et al. J. Virol. 61:3688-3693(1987)), which does not recognize endogenous wt MoPrP in ScN2a cells andhence facilitates specific detection of the transgene by Western blot.

[0275] Following electrophoresis, Western blotting was performed aspreviously described (Scott et al. Cell 59:847-857 (1989)). Samples wereboiled for 5 minutes and cleared by centrifugation for 1 minute at14,000 rpm in a Beckman ultrafuge. SDS-PAGE was carried out in 1.5 mm,12% polyacrylamide gels (Laemmli (1970) Nature 227:661-665). Membraneswere blocked with 5% nonfat milk protein in PBST (calcium- andmagnesium-free PBS plus 0.1% Tween 20) for 1 hour at room temperature.Blocked membranes were incubated with primary RO73 polyclonal or 3F4monoclonal antibody at a 1:5000 dilution in PBST overnight at 4° C.

[0276] Following incubation with primary antibody, membranes were washed3×10 minutes in PBST, incubated with horseradish peroxidase-labeledsecondary antibody (Amersham Life Sciences) diluted 1:5000 in PBST for25 minutes at room temperature and washed again for 3×10 minutes inPBST. After chemiluminescent development with ECL reagent (Amersham) for1 minute, blots were sealed in plastic covers and exposed to ECLHypermax film (Amersham). Films were processed automatically in a Konicafilm processor.

[0277] In contrast to DOTAP-transfected cells, ScN2a cells transfectedwith varying concentrations of SuperFect™ and DNA did not appear tocontain protease-resistant MHM2. Close scrutiny revealed that, prior toprotease digestion, SuperFect™-transfected samples express MHM2 bands,which are not seen in the background pattern of the control sample.These observations indicate that MHM2 PrP was successfully expressedusing SuperFect™ transfection reagent, but conversion of MHM2 PrP^(C) toprotease-resistant MHM2 PrP^(Sc) was inhibited by SuperFect™.

[0278] To examine whether SuperFect™ had affected levels of preexistingPrP^(Sc) in ScN2a cells, the Western blot probed with 3F4 antibody wasreprobed with polyclonal antibody RO73, which is able to recognizeendogenous MoPrP. Remarkably, SuperFect™ caused the disappearance ofpreexisting MoPrP^(Sc) from ScN2a cells in a dose-dependent manner.After treatment with SuperFect™, PrP^(Sc) could not be detected in thenuclear fraction, pellet, supernatant, or media. The concentration ofSuperFect™ required to fully remove preexisting PrP^(Sc) with athree-hour exposure was 300 μg/ml, whereas 30 μg/ml was sufficient tointerfere with the formation of new MHM2 PrP^(Sc) within the same timeframe.

[0279] Length of exposure dramatically influenced the ability ofSuperFect™ to remove Prp^(Sc) from ScN2a cells. Whereas a three-hourexposure to 150 μg/ml SuperFect™ significantly lowered PrP^(Sc) levelsin ScN2a cells, exposure for 10 min to the same dose of SuperFect™ didnot affect PrP^(Sc) levels. When ScN2a cells were exposed to 2 μg/mlSuperFect™ continuously for 1 week, PrP^(Sc) disappeared completely.

[0280] The conditions tested did not appear to be toxic for the cells.Neither 150 μg/ml SuperFect™ for 3 hrs nor 2 μg/ml SuperFect™continuously for 1 week caused any obvious changes in cell morphology,viability, or growth as judged by phase contrast microscopy.

Example 7 Elimination of PrP^(Sc) by Repeated Exposures to SuperFect™

[0281] The duration in the reduction in PrP^(Sc) levels after exposureto SuperFect™ was examined, and it was shown that this reduction couldpersist for extended periods after removal of SuperFect™. Following theexposure of ScN2a cells to a single dose of 150 μg/ml SuperFect™ for 3hrs, PrP^(Sc) levels remained low for one week, but returned to nearbaseline levels after 3 weeks in culture without SuperFect™.

[0282] In contrast, when ScN2a cells were exposed to 4 separate doses ofSuperFect™ over the course of 16 days, very little PrP^(Sc) could bedetected 4 weeks after the final exposure to SuperFect™. This resultoffers hope that prolonged exposure to SuperFect™ may lead to long termcure of scrapie infection in cultured cells.

Example 8 SuperFect™ Does Not Destroy PrP^(Sc) Directly

[0283] The dendrimer SuperFect™ was used to determine if it could exerta similar inhibitory effect on PrP^(Sc) in either crude brainhomogenates or purified PrP 27-30 rods. Brain homogenates from normaland scrapie-affected Syrian hamsters (10% (w/v) in sterile PBS) wereprepared by repeated extrusion through syringe needles of successivelysmaller size, from 18 to 22 gauge. Nuclei and debris were removed bycentrifugation at 1000×g for 10 min. The bicinchnoninic acid (BCA)protein assay (Pierce) was used to determine protein concentration.Homogenates were adjusted to 10 mg/ml protein with PBS and 50 μl wasadded to 450 μl of lysis buffer containing 100 mM NaCl, 1 mM EDTA, 0.55%sodium deoxycholate, 0.55% Triton X-100, and 50 mM Tris-HCl pH 7.5. Thismixture was then incubated with 0-300 μg/ml SuperFect™ for 3 hrs at 37°C. and then centrifuged for 10 min at 14,000 rpm in a Beckman Ultrafuge.The pellet was resuspended in 450 μl lysis buffer without SuperFect™.Proteinase K (Boehringer Mannheim) was added to achieve a finalconcentration of 20 μg/ml, and thus the ratio of total protein/enzymewas 50:1. Samples were incubated for 1 h at 37° C. Proteolytic digestionwas terminated by the addition of 8 μl of 0.5 M PMSF in ethanol. Sampleswere then centrifuged for 75 min in a Beckman TLA-45 rotor at 100,000×gat 4° C. Undigested samples (10 μl) were mixed with an equal volume of2×SDS sample buffer. For digested samples, the pellet was resuspended byrepeated pipetting in 100 μl 1×SDS sample buffer. Twenty μl (equivalentto 100 μg of total protein prior to proteinase K digestion) of eachsample was loaded for SDS-PAGE.

[0284] PrP 27-30 rods were purified from scrapie-affected Syrian hamsterbrains and previously described (Prusiner et al. Cell 35:349-358(1983)). Purified rods (3.5 μg/ml) were incubated with or without 900μg/ml SuperFect™ in 100 μl supplemented DME. After 16 hrs at 37° C., thesuspension was centrifuged at 100,000×g at 4° C. The pellet wasresuspended in 500 μl of buffer containing 1 mg/ml BSA, 100 mM NaCl, 1mM EDTA, 0.55% sodium deoxycholate, 0.55% Triton X-100, and 50 mMTris-HCl pH 7.5. Proteinase K was added to achieve a final concentrationof 20 μg/ml. Samples were incubated for 1 h at 37° C. Proteolyticdigestion was terminated by the addition of 8 μl of 0.5 M Pefabloc(Boehringer Mannheim). Samples were then centrifuged for 75 min at100,000×g at 4° C. Undigested samples (50 μl) were mixed with an equalvolume of 2×SDS sample buffer. For digested samples, the pellet wasresuspended by repeated pipetting in 100 μl 1×SDS sample buffer. Fortyμl of each sample was loaded for SDS-PAGE.

[0285] When SuperFect™ was mixed with either crude homogenates ofscrapie-affected Syrian hamsters or with purified Syrian hamster PrP27-30, there was no significant change in the level of proteinaseK-resistant PrP^(Sc). These results suggest that the removal of PrP^(Sc)from ScN2a cells by SuperFect™ depends on the presence of intactcellular machinery.

Example 9 Clearance of PrP^(Sc) Levels by Other Dendritic Polycations

[0286] The SuperFect™ compound is a high molecular weight component ofheat-degraded PAMAM Starburst dendrimers, which is a cationic,highly-branched, monodisperse polymers (Tang et al., Bioconjugate Chem.7:703-714 (1996)). To identify other potentially useful anti-priontherapeutic agents, three other dendritic polycations and two linearcationic polymers were screened for their ability to clear PrP^(Sc) fromScN2a cells. Among the dendritic macromolecules tested,polyetheleneimine (PEI) was the most potent, removing the majority ofPrP^(Sc) from ScN2a cells after 3 hrs when used at a concentration of 10μg/ml. Intact PAMAM displayed a potency comparable to SuperFect™,removing approximately half of the detectable PrP^(Sc) when used at aconcentration of 50 μg/ml. In contrast, the dendrimer polypropyleneimine(PPI), poly-(L)lysine, and the linear polycation poly-(D)lysine failedto reduce PrP^(Sc) levels at concentrations between 10-50 μg/ml. Theseresults demonstrate that a branched polymeric architecture is requiredto clear PrP^(Sc). Furthermore, exposure of ScN2a cells to either PEI orintact PAMAM for one week at a concentration of 1.5 μg/ml completelyremoves PrP^(Sc), effectively curing the cells of scrapie infection.

Example 10 Branched Polyamines Cure Prion-Infected Neuroblastoma Cells

[0287] The above Examples show that branched polyamines purgedscrapie-infected neuroblastoma (ScN2a) cells of PrP^(Sc), theprotease-resistant isoform of the prion protein. The ability of thesecompounds to eliminate PrP^(Sc) from ScN2a cells depended upon certainmolecular characteristics. In particular, active compounds were highlybranched and possessed a high surface density of primary amino groups.The most potent compounds identified were generation 4.0 polyamidoamide(PAMAM) and polypropyleneimine (PPI) dendrimers. Dendrimers are branchedpolyamines manufactured by a repetitive divergent growth technique,allowing the synthesis of successive, well-defined “generations” ofhomodisperse structures. The following experimental results demonstratethat branched polyamines cure prion-infected cells. The site andmechanism of action for these compounds was also determined.

[0288] Materials and Methods

[0289] Chemical compounds. High molecular weight PEI was purchased fromFluka. SuperFect™ transfection reagent was purchased from QIAGEN®. Allother polyamines were purchased from Sigma-Aldrich. Fluorescein-labeledPPI was synthesized as follows: 30 mg fluorescein isothiocyanate (FITC)was mixed with 1 mg PPI generation 4.0 in 2 ml absolute ethanolovernight at 4° C. A 3:1 excess of PPI-to-FITC equivalent groups was setup to minimize the production of multiple FITC conjugates per PPI.Labeled PPI was separated from residual, unreacted FITC using a 12 mm×37cm Sephadex P-2 column equilibrated in 0.15 mM NaCl buffer. Fractionswere collected and analyzed by thin layer chromatography for singlespots of fluorescence and amine content. Fluorescence was detected usinga long wave UV lamp, and primary amines were detected by ninhydrinassay. Appropriate fractions were combined and lyophilized. The drypowder was brought up in sterile water, titrated to pH 7.0, diluted in5% glucose, 5 mM HEPES pH 7.4, and filtered through a 0.2 Fmpolycarbonate membrane. FITC concentration of this stock solution was44.1 FM, as measured by UV spectroscopy with an absorbance maxima at 489nm. Final PPI concentration was 50 FM.

[0290] Cultured Cells

[0291] Cultures of ScN2a cells were maintained in DME pH 7.4 with 10%FBS, 10% Glutamax (Gibco BRL), 100 U/ml penicillin, and 100 Fg/mlstreptomycin (supplemented DME). Cultures were split 1:10 weekly, andfed fresh medium twice weekly. Cytotoxicity after treatment withpolyamines was assessed in ScN2a cells by four methods: (1) examinationof morphology under phase contrast microscopy, (2) observation of growthcurves and cell counts for three weeks after treatment, (3) vitalstaining of living cells with 0.4% trypan blue (Sigma-Aldrich), and (4)assay of dehyrogenase enzymes with93-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Forthe dehyrogenase assay, cells in 96-well plates were incubated with 0.5mg/ml MTT (Sigma-Aldrich) in supplemented DME for 4 hrs. Media was thenaspirated and cells were dissolved in isopropanol containing 50 mM HCl.Converted MTT was measured by absorbance at 570 nm. For ScN2a cellstreated with either PAMAM or PPI generation 4.0 continuously for 1 week,LD₅₀˜50 Fg/ml.

[0292] To prepare samples for infectivity assays, 100 mm plates (Falcon)of confluent cells were washed with 3×5 ml PBS, scraped into 2 ml PBS,and homogenized by repeated extrusion through a 26 gauge needle. Prioninfectivity was determined by intracerebral inoculation of 30 Fl cellhomogenate into Tg(MoPrP)4053 mice. Mice were observed for clinicalsigns of scrapie, and a subset of diagnoses were confirmed byneuropathological examination.

[0293] To prepare samples for SDS-PAGE, plates were drained of media andadherent cells were lysed in 1 ml 20 mM Tris pH 8.0, 100 mM NaCl, 0:5%NP-40, 0.5% sodium deoxycholate. Samples were adjusted to obtain a totalprotein concentration of 1 mg/ml measured by the bicinchnoninic (BCA)assay (Pierce). Nuclei were removed from the lysate by centrifugation at2000 rpm for 5 min. For samples not treated with proteinase K, 10 Fl oflysate was mixed with an equal volume of 2×SDS reducing sample buffer.For proteinase K digestion, 20 Fg/ml proteinase K (Boehringer Mannheim)(total protein:enzyme ratio 50:1) was incubated with 1 ml lysate for 1hr at 37° C. Proteolytic digestion was terminated by the additionPefabloc (Boehringer Mannheim) to a final concentration of 5 mM. Sampleswere then centrifuged at 100,000×g for 1 hr at 4° C. and the pelletfractions were resuspended in 80 Fl of reducing SDS sample buffer.Twenty microliter samples were loaded per lane on 1 mm 12% Tris glycineSDS-PAGE gels (Novex).

[0294] Mixture of brain homogenates and purified prions with polyaminesin vitro. Brain homogenates from scrapie-infected rodents (10% (w/v) insterile water) were prepared by repeated extrusion through syringeneedles of successively smaller size, from 18 to 22 gauge. Nuclei anddebris were removed by centrifugation at 1000×g for 5 min. Homogenateswere adjusted to 1 mg/ml protein in 1% NP-40. For incubations with PPI,50 Fl 1 mg/ml brain homogenate was mixed with 450 Fl 1% NP40, 50 mMsodium acetate pH 3.0 (final measured pH=3.6) plus or minus 60 Fg/ml PPIgeneration 4.0 and shaken constantly for various periods at 37° C.

[0295] Purified prions were prepared as described previously, utilizingboth proteinase K digestion and sucrose gradient sedimentation, andresuspended in 1% NP-40, 1 mg/ml BSA. For pH studies, 475 Fl of 0.5Fg/ml purified RML PrP27-30 in 1% NP-40, 1 mg/ml BSA was mixed with 25Fl 1M buffers from pH 3-8 (sodium acetate for pH 3-6 and Tris acetatefor pH 7-8) plus or minus 60 Fg/ml PPI generation 4.0 for 2 hrs at 37°C. with constant shaking. The final pH value of each sample was measureddirectly with a calibrated pH electrode (Radiometer Copenhagen). Forcompound screening, 475 Fl of 0.5 Fg/ml purified RML PrP27-30 in 1%NP-40, 1 mg/ml BSA was mixed with 25 Fl 1M sodium acetate pH 3.0 plus 60Fg/ml polyamine for 2 hrs at 37° C. with constant shaking.

[0296] Following incubations, each sample was neutralized with an equalvolume 0.2 M HEPES pH 7.5 containing 0.3 M NaCl and 4% Sarkosyl. Samplesnot treated with proteinase K were mixed with equal volume 2×SDS samplebuffer. For proteinase K digestion, samples were incubated with 20 Fg/mlproteinase K (Boehringer Mannheim) (total protein:enzyme ratio 25:1) for1 hr at 37° C. Proteolytic digestion was terminated by the addition of 8μl of 0.5M PMSF in absolute ethanol. Digested samples were then mixedwith equal volumes 2×SDS sample buffer. All samples were boiled for 5min prior to electrophoresis. SDS-PAGE was performed on 1.5 mm 12%polyacrylamide gels.

[0297] Western blotting. Following electrophoresis, Western blotting wasperformed as previously described. Membranes were blocked with 5%non-fat milk protein in PBST (calcium- and magnesium-free PBS plus 0.1%Tween 20) for 1 hr at room temperature. Blocked membranes were incubatedwith 1 Fg/ml recombinant, humanized Fab d13 in PBST for 1 hr at 4° C.Following incubation with primary Fab d13, membranes were washed 3×10min in PBST, incubated with horseradish peroxidase-labeled anti-humanFab secondary antibody (ICN) diluted 1:5000 in PBST for 45 min at roomtemperature and washed again for 4×10 min in PBST. Afterchemiluminescent development with ECL reagent (Amersham) for 1-5 min,blots were sealed in plastic covers and exposed to ECL Hypermax film(Amersham). Films were processed automatically in a Konica filmprocessor.

[0298] Negative stain electron microscopy. Sample preparation was doneon carbon-coated 600 mesh copper grids that were glow-discharged for 30sec prior to staining. Five microliter samples were adsorbed to gridsfor 30-60 sec, washed with 2 drops of 0.1 M and 0.01 M ammonium acetateeach, and stained with 2 drops of freshly filtered 2% ammonium molybdateor uranyl acetate. After drying, samples were viewed in a Jeol JEM 100CXII electron microscope at 80 kV at a standard magnification of 40,000.The magnification was calibrated using negatively-stained catalasecrystals.

[0299] Confocal microscopy. Confocal images were obtained using a BioRadlaser scanning confocal microscope (MRC-1024, Hercules, Calif.),outfitted with a Nikon Diaphot 200 microscope and a Helium/Neon laser. A60×Nikon planAPO lens was used, with an additional software zoomfunction. Laser power was set at 10%, and scanned with a slow speedacross the sample. Individual laser lines confirmed the lack of “bleedthrough” between detection channels. The images were averaged with aKalman filter (n=4).

[0300] ScN2a cells were incubated with 3 Fg/ml PPI in supplemented DMEfor 4 weeks, and then cultured for an additional 2 weeks inpolyamine-free medium. This transient exposure to PPI was not cytotoxic(see Methods) and completely purged the cells of protease-resistantPrP^(Sc) (FIG. 2A, lanes 2 and 4). In contrast, protease-sensitivePrP^(C) bands migrating between 32-38 kDa appear unaltered by PPItreatment (lanes 1 and 3).

[0301] Elimination of PrP^(Sc) appeared to be relatively specific sincethe steady-state levels of proteins in PPI-treated cells was similar tothose in control ScN2a cells (FIG. 2B). To assess the effect of PPItreatment on prion infectivity, homogenates prepared frompolyamine-treated and control ScN2a cells were inoculated intoTg(MoPrP)4053 mice. The average scrapie incubation time was 67+2 daysfor mice inoculated with control ScN2a cells and >120 days for miceinoculated with ScN2a cells treated with PPI (n/n₀=0/10) (FIG. 2C).These incubation times indicate that the titer of infectious prions inScN2a cells was reduced from ˜10⁷ LD₅₀ units/100 mm plate to <1 LD₅₀unit/plate by PPI treatment. Thus, exposure to PPI completely eliminatesprion infectivity from ScN2a cells.

[0302] Treatment of scrapie-infected neuroblastoma cells withpolyamidoamide dendrimer. ScN2a cells were treated with 3 Fg/ml PPIgeneration 4.0 in supplemented DME or control media for 4 weeks. After 2additional weeks of culture in compound-free media, cells were harvestedfor analysis. (FIG. 2A) PrP immunostain with recombinant Fab d13 wasperformed as described in the Methods section. Apparent molecularweights based on migration of protein standards are 30 and 27 kDa. (FIG.2B) Silver stain was performed as previously described. Apparentmolecular weights based on migration of protein standards are 49, 36,25, and 19 kDa. (FIGS. 2A and 2B) Samples subjected to SDS-PAGE wereassigned lanes as follows: (FIG. 2A, lanes 1 and 3) control cells, (FIG.2A, lanes 2 and 4) PPI-treated cells. Lanes (1) and (2) containundigested lysates, and lanes (3) and (4) contain lysates subjected tolimited proteolysis with proteinase K. FIG. 2C is an infectivitybioassay of cell homogenates in Tg(MoPrP)4053 indicator mice: (Filledcircles) control cells, (Open squares) PPI-treated cells.

Example 11 Branched Polyamines Act Directly on Purified RML Prions

[0303] Because branched polyamines reduce prion infectivity, the nextstep was to determine the mechanism by which these compounds eliminatePrP^(Sc). An initial objective was to determine the molecular target ofbranched polyamines. A previously described in vitro assay was used toshow that these compounds could render PrP^(Sc) protease-susceptiblewhen mixed directly with crude brain homogenates. A similar assay wascarried out with purified RML PrP27-30 prions to determine whether ornot the molecular target of branched polyamines was present in thishighly purified preparation. PrP^(Sc) in purified preparations of RMLPrP27-30 were rendered protease-sensitive by branched polyamines with asimilar acidic pH optimum (FIG. 3A) and structure-activity profile (FIG.3B) as previously obtained in crude brain homogenates. These resultsindicate that the molecular target of branched polyamines must eitherbe: (1) PrP^(Sc) itself, (2) an acid-induced unfolding intermediate ofPrP^(Sc); or (3) a very tightly bound, cryptic molecule which copurifieswith PrP^(Sc).

[0304]FIGS. 3A and B show a mixture of purified prions with branchedpolyamines in vitro. FIG. 3A shows, samples containing 0.5 Fg/mlpurified mouse RML PrP27-30 in 1 mg/ml BSA incubated for 2 hrs at 37° C.with 60 Fg/ml PPI generation 4.0 or control buffer at different pHvalues as indicated. FIG. 3B shows samples containing 0.5 Fg/ml purifiedmouse RML PrP27-30 in 1 mg/ml BSA incubated for 2 hrs at 37° C. withvarious polyamines: (lanes 1-2) control, (lane 3) poly-(L)lysine, (lane4) PAMAM 0.0, (lane 5) PAMAM 1.0, (lane 6) PAMAM 2.0, (lane 7) PAMAM3.0, (lane 8) PAMAM 4.0, (lane 9) PAMAM-OH 4.0, (lane 10) PPI 2.0, (laneI) PPI 4.0, (lane 12) linear PEI, (lane 13) high MW PEI, (lane 14) lowMW PEI, (lane 15) average MW PEI, (lane 16) Qiagen SuperFect. Allcompounds were tested at a concentration of 60 Fg/ml. Westernimmunoblotting was performed with recombinant Fab d13 for both FIGS. 3Aand 3B. Apparent molecular weights based on migration of proteinstandards are 30 and 27 kDa.

Example 12 PrP^(Sc) Susceptibility to PPI-Induced Conformational Changeis Sequence- and Strain-Specific

[0305] Although the PrP sequence is well conserved among mammals, asmall number of amino acid substitutions appear to hinder priontransmission across species. Furthermore, prions can exist as differentphenotypic strains that yield distinct incubation times, neuropathology,and distribution of PrP^(Sc) upon infection of susceptible hosts. Incertain cases, these phenotypic differences can be correlated withdifferences in the conformation of PrP^(Sc). An effort was made todetermine whether different species and strains of rodent prions, whichpresumably contain different conformations of PrP^(Sc), vary in theirsusceptibility to branched polyamines. Homogenates were prepared fromthe brains of rodents infected with one of several Syrian hamster (SHa),mouse (Mo), or artificial prion strains. Individual samples were mixedwith 60 Fg/ml PPI generation 4.0 in vitro for 2 h at 37° C.,neutralized, and subjected to limited proteolysis. The results indicatethat susceptibility to PPI dendrimer is dependent on both prion strainand PrP sequence (FIG. 4A).

[0306] The varying susceptibility of different strains is most clearlyillustrated by the 6 mouse strains analyzed (paired lanes 7-12).Mo(RML), Mo(22a), and Mo(139A) were susceptible to PPI-inducedconformational change (paired lanes 7, 9, and 11, respectively). Incontrast, Mo(Me7) and Mo(87V) were resistant (paired lanes 8 and 10,respectively); and Mo(C506) was marginally susceptible to PPI-inducedconformational change (paired lane 12).

[0307] The effect of PrP sequence can be seen by comparing the relativesusceptibilities of SHa(RML), MH2M(RML), and Mo(RML). Whereas Mo(RML)was susceptible to PPI-induced conformational change (paired lane 7),SHa(RML) was resistant (paired lane 4). MHM2(RML) displayed anintermediate level of susceptibility to PPI (paired lane 5); MHM2 is achimeric PrP molecule in which amino acids 94 to 188 of the mousesequence have been replaced by the corresponding SHa residues. Thus,SHaPrP^(Sc) appears to be more resistant to PPI-induced conformationalchange than MoPrP^(Sc).

[0308] The varying susceptibilities to PPI displayed by differentstrains and species of prions, which might be caused by kineticdifferences, was investigated. To test this possibility, samples of eachprion isolate were incubated with PPI generation 4.0 for varying periodsof time. Even after incubation with PPI for 3 days, PrP^(Sc) in samplesof resistant isolates did not become more susceptible to proteasedigestion (for example, FIG. 4B). Thus, the differences insusceptibilities of different prion strains and sequences are not causedsimply by differences in the kinetics of PrP^(Sc) unfolding.

[0309] The results shown in FIG. 4 show the treatment of different prionstrains with polypropyleneimine in vitro (FIG. 4A). Samples containing1% (w/v) various brain homogenates were incubated for 2 hrs at 37° C.with 60 Fg/ml PPI generation 4.0. Paired lanes are designated asfollows: (lane 1) SHa(Sc237), (lane 2) SHa(139H), (lane 3) SHa(drowsy),(lane 4) SHa(RML), (lane 5) Tg(MH2M)Prnp^(0/0)(RML), (lane 6 )Tg(PrP106) Prnp^(0/0)(RML), (lane 7) mouse(RML), (lane 8) mouse(Me7),(lane 9) mouse(22a), (lane 10) mouse(87V), (lane 11) mouse(139A), (lane12) mouse(C506). Minus (−) symbol denotes undigested, control sample andplus (+) symbol designates sample subjected to limited proteolysis byproteinase K.

[0310]FIG. 4B shows samples containing 1% (w/v) mouse(RML) or SHa(Sc237)incubated at 37° C. with 60 Fg/ml PPI generation 4.0 or control bufferfor the time periods indicated. Western immunoblotting was performedwith recombinant Fab d13 for both FIGS. 4A and 4B. Apparent molecularweights based on migration of protein standards are 30 and 27 kDa. Prionstrains were obtained from the following sources: SHa(Sc237), Mo(139A),and Mo(Me7) from R. Kimberlin; SHa(139H) from R. Carp; SHa(drowsy) fromR. Marsh; Mo(RML) from W. Hadlow; Mo(22a) and Mo(87V) from A. Dickenson;Mo(C506) from J. Gibbs. SHa(RML) was obtained by passaging Mo(RML)directly into Syrian hamsters. The passage histories of other strainshave been reviewed (Ridley, 1996).

Example 13 Branched Polyamines Assist PrP^(Sc) Disaggregation

[0311] The existence of prion strains resistant to branched polyaminessuggests that PrP^(Sc) molecules in these strains might exist inconformations which are more resistant to denaturation than PrP^(Sc)molecules in polyamine-susceptible strains. To test this hypothesis, anexamination was made of the effect of adding urea to SHa(Sc237) brainhomogenate treated with and without PPI generation 4.0 . In the presenceof urea, PrP^(Sc) was more susceptible to protease digestion in samplestreated with PPI, whereas no difference in protease-resistance could bedetected in the absence of urea (FIG. 5A). Thus, additional denaturationenables PrP^(Sc) molecules in a resistant strain to become susceptibleto branched polyamines. This result suggests that the general mechanismof action of branched polyamines might be to assist PrP^(Sc)denaturation. Consistent with this concept, branched polyamines renderPrP^(Sc) protease-sensitive more efficiently at lower pH values (FIG.4A) and higher temperatures (FIG. 5B). Furthermore, polyamine-treatedPrP^(Sc) did not regain protease-resistance after prolongedneutralization (FIG. 5C) or dialysis (data not shown). Finally, thepossibility was excluded that acidification might be required only toactivate the dendrimer. It was demonstrated that pre-acidified PPIgeneration 4.0 could not render PrP^(Sc) protease-sensitive at neutralpH FIG. 5D).

[0312] To visualize the effect of branched polyamines on prions, theultrastructure of purified prion rods treated in vitro with PPIgeneration 4.0 was examined. By electron microscopy, Mo(RML) PrP27-30rods were disaggregated after incubation for 2 hrs at 37° C. with PPI(FIG. 6B). In contrast, SHa(237) PrP27-30 rods remained intact aftertreatment with PPI (FIG. 6D).

[0313] To investigate further the mechanism of polyamine-induceddisaggregation of PrP^(Sc), a kinetic study in vitro was performed usingpurified RML PrP27-30 and various concentrations of PPI. The resultsindicate that polyamine-induced PrP^(Sc) disaggregation is not acatalytic process, and requires a stoichiometry of approximately 1 PPImolecule per 5 RML PrP27-30 molecules in purified prion preparations.

[0314] Denaturation of PrP^(Sc) is enhanced by polypropyleneimine, asshown in FIG. 5A. Samples containing 1% (w/v) SHa(Sc237) brainhomogenates were incubated for 2 hrs at 37° C. with 60 Fg/ml PPIgeneration 4.0 or control buffer, plus various concentrations of urea asindicated. All samples were subjected to limited proteolysis. FIG. 5Bshows samples containing 0.5 Fg/ml purified mouse RML PrP27-30 in 1mg/ml BSA incubated for 2 hrs at various temperatures with 60 Fg/ml PPIgeneration 4.0 . Paired lanes are designated as follows: (lane 1) 4° C.(lane 2) 20° C., (lane 3) 37° C. Minus (−) symbol denotes undigested,control sample and plus (+) symbol designates sample subjected tolimited proteolysis.

[0315]FIG. 5C shows samples containing 1% mouse(RML) brain homogenate in1% NP40, 50 mM sodium acetate pH 3.6 incubated at 37° C. for 2 hrs witheither: (odd lanes) no addition or (even lanes) 60 Fg/ml PPI. Allsamples were neutralized with an equal volume 0.2 M HEPES pH 7.5containing 0.3 M NaCl and 4% Sarkosyl. Lanes 1 and 2 samples notsubjected to protease digestion, lanes 3 and 4 samples immediatelysubjected to limited proteinase K digestion, lanes 5 and 6 samplesincubated at pH 7.5 for an additional 16 hrs at 37° C. before proteinaseK digestion.

[0316]FIG. 5D shows samples containing 1% mouse(RML) brain homogenatetreated in the following manner: (lane 1) control sample at pH 3.6,(lane 2) mixed with 60 Fg/ml PPI at pH 3.6 for 2 hrs, (lane 3) mixedwith 60 Fg/ml PPI at pH 7.0 for 2 hrs, (lane 4) incubated alone at pH3.6 for 2 hrs and then mixed with 60 Fg/ml PPI (pre-titrated to pH 7.0)for 10 min, (lane 5) incubated alone at pH 7.0 for 2 hrs and then mixedwith 60 Fg/ml PPI (pre-titrated to pH 3.0) for 10 min. All incubationswere carried out at 37° C. Minus (−) symbol denotes undigested, controlsample and plus (+) symbol designates sample subjected to limitedproteolysis by proteinase K. Western immunoblotting was performed withrecombinant Fab d13 for FIGS. 5A-D. Apparent molecular weights based onmigration of protein standards are 30 and 27 kDa.

[0317]FIG. 6 shows the ultrastructure of purified prion rods treatedwith polyproplyeneimine in vitro. Samples of purified 100 Fg/ml PrP27-30in 0.1% NP40, 50 mM sodium acetate pH 3.0 buffer were shakencontinuously for 2 hrs at 37° C. Samples were then prepared for negativestain electron microscopy as described in Methods. FIG. 6A is mouse(RML)plus 60 Fg/ml PPI generation 4.0 , and FIG. 6B is SHa(Sc237) plus 60Fg/ml PPI generation 4.0 . Negative stain used was 2% uranyl acetate;scale bar=100 nm.

Example 14 PPI Accumulates in Lysosomes

[0318] Branched polyamines apparently require acidic conditions torender PrP^(Sc) protease-sensitive when mixed with brain homogenates orpurified prions in vitro, as shown in FIG. 3A. However, these compoundssuccessfully cure living culture media buffered at pH 7.4, as shown inFIG. 2. One possible explanation for this discrepancy is that branchedpolyamines might co-localize with prions within an acidic intracellularcompartment. PrP^(Sc) has previously been shown to accumulate inlysosomes. Therefore, it was investigated whether branched polyamineslocalize to this same compartment. N2a cells were incubated withfluorescein-labeled PPI and LysoTracker Red, and performed dual channelconfocal microscopy to compare the localization of the two compounds.The results indicate that fluorescein-labeled PPI accumulates in thelysosomes of living cells (FIGS. 7A, 7B and 7C).

[0319] Polypropyleneimine localizes to lysosomes N2a cells grown to 50%confluence on coverslips were incubated for 4 hrs with 3 FM FITC-PPI and1 hr with 75 nM LysoTracker Red (Molecular Probes) in supplemented DME.Following incubation, coverslips were washed 3 times with PBS, fixedwith 2% paraformaldehyde in PBS, and mounted onto microscope slides withVECTASHIELD. Fluorescence confocal microscopy in green and red channelswas performed as described in Methods. Scale bar=5 Fm.

[0320] Branched Polyamines as Therapeutic Agents

[0321] A major finding of the above Examples is that branched polyamineseliminate prion infectivity in chronically infected living cells. Thisis believed to be the first class of compounds shown to cure anestablished prion infection. Polyene antibiotics, anionic dyes,sulphated dextrans, anthracylines, porphyrins, phthalocyanines, dapsone,and a synthetic β-breaker peptide all prolong scrapie incubation timesin vivo, but only if administered very early in the course of infection.

[0322] The unique ability of branched polyamines to cure an establishedprion infection in cells indicates that they can be used for a therapyin animals, even when administered after the onset of symptoms. However,two factors could potentially limit the use of these compounds astherapeutic agents against prion diseases. Branched polyamines might notact on all strains of prions, and they might not cross the blood-brainbarrier. The first possibility is suggested by our data, which show thatsome strains and species of prions are more resistant than others tobranched polyamine-induced disaggregation in vitro. It remains to bedetermined whether prion strains resistant to branched polyamine-induceddisaggregation in vitro would also be resistant to treatment by thesecompounds in vivo. Treatment of more resistant strains might requiretherapy with branched polyamines in combination with another class ofprion-directed compounds.

[0323] The second potential limitation of branched polyamines is thatthese highly charged compounds might not cross the blood-brain barrier.If this proves to be the case, branched polyamines could be delivereddirectly to the CSF through an intraventricular reservoir, or perhapssynthesized as prodrugs capable of crossing the blood-brain barrier.Preliminary studies indicate that continuous intraventricular infusionof PPI generation 4.0 is tolerated by FVB mice up to a total dose ofapproximately 2 mg/animal (data not shown).

[0324] Molecular Target, Mechanism, and Site of Action

[0325] It is important to characterize the molecular and cellularmechanisms by which branched polyamines eliminate prions for tworeasons. First, branched polyamines could potentially be used asresearch tools to study the cellular and structural biology of prions.Second, identifying the molecular target of branched polyamines wouldfacilitate the design of other compounds more specifically directedagainst this target.

[0326] The ability of branched polyamines to render PrP^(Sc)protease-sensitive in purified preparations, as shown in FIGS. 3A and3B, suggests that the molecular target of these compounds must eitherbe: (1) PrP^(Sc) itself, (2) an acid-induced unfolding intermediate ofPrP^(Sc); or (3) a very tightly bound, cryptic molecule which copurifieswith PrP^(Sc). If the molecular target is PrP, at least one of thepolyamine binding sites must be contained within the amino acid sequenceof the PrP106 deletion mutant, since PPI renders PrP^(Sc) 106protease-sensitive (FIG. 4A, lane 6 ). The 106 amino acids present inPrP106 are residues 89-140 and 177-231. PPI also renders a spontaneouslyprotease-resistant, 61 amino acid-long PrP deletion mutant, PrP()23-88,)141-221), susceptible to protease-digestion, further confiningthe boundaries of at least one putative binding site to residues 89-140and 222-231.

[0327] Several lines of evidence suggest that branched polyamines renderPrP^(Sc) molecules protease-sensitive by dissociating PrP^(Sc)aggregates. (1) RML PrP27-30 prion rods treated in vitro with PPI becomedisaggregated, as judged by electron microscopy (FIGS. 6A and 6B). (2)Prion strains resistant to branched polyamines in vitro appear to bemore amyloidogenic than polyamine-susceptible strains, as judged byneuropathology. (3) The ability of branched polyamines to renderPrP^(Sc) protease-sensitive in vitro is enhanced by conditions thatfavor PrP^(Sc) disaggregation. These conditions include lower pH (FIG.3A), higher temperature (FIG. 5B), and the presence of urea (FIG. 5A).

[0328] Theoretically, it is possible that the mechanism by whichbranched polyamines remove PrP^(Sc) and prion infectivity from ScN2acells does not relate to the ability of these compounds to disaggregateprions in vitro. However, this is unlikely because the relative potencyof 14 different polyamines in eliminating PrP^(Sc) from ScN2a cellsexactly matches the relative ability of these same compounds to renderPrP^(Sc) protease-sensitive in crude brain homogenates and purifiedpreparations of RML PrP27-30 in vitro (FIG. 3B). The structure-activityprofile obtained from these studies indicates that polyamines becomemore potent at eliminating PrP^(Sc) as they become more branched andpossess more surface primary amines. With PPI dendrimers, this effectreaches a plateau at the fourth generation; PPI generation 5.0 is nomore potent than PPI generation 4.0 at either removing PrP^(Sc) fromcells or rendering PrP protease-sensitive in vitro. Homodisperse,uniform PPI and PAMAM dendrimers were more potent than the heterogeneouspreparations of polyethyleneimine (PEI) or SuperFect™, a heat-fractureddendrimer:

[0329] The process by which PPI renders PrP^(Sc) protease-sensitive invitro was not catalytic. Instead, this process appeared to require afixed stoichiometric ratio of PPI to PrP^(Sc) of approximately 1:5. Thequestion was presented regarding how PPI could disaggregate prion rodsstoichiometrically. One possible explanation is that individual aminogroups on the surface of PPI might bind to PrP^(Sc) monomers oroligomers that exist in equilibrium with a large aggregate under acidicconditions. The dendrimer might then pry bound PrP^(Sc) molecules apartfrom the aggregate and/or prevent such molecules from reaggregating.

[0330] Several lines of evidence indicate that the cellular site ofaction of branched polyamines is secondary lysosomes. (1)Fluorescein-tagged PPI and PrP^(Sc) both localize to lysosomes (FIGS.7A-C). (2) The pH optimum of PrP^(Sc) disaggregation in vitro is <5.0.When cultured cells were studied with fluorescent acidotroptic pHmeasurement dyes, secondary lysosomes were the most acidic cellularcompartment detected, with pH values of ˜4.4-4.5. (3) The lysosomotropicagent chloroquine attenuates the ability of branched polyamines toeliminate PrP^(Sc) from ScNa cells. It is thought that lysosomalproteases normally degrade PrP^(Sc) in prion-infected cells at a slowrate, and that polyamines accelerate this process by disaggregatingPrP^(Sc).

[0331] Other Applications of Branched Polyamines

[0332] Beyond their potential use as therapeutic agents and researchtools, branched polyamines might also be useful as prion strain typingreagents and/or prion decontaminants. Presently, typing of prion strainsis time-consuming and requires the inoculation of samples into severalstrains of inbred animals to obtain incubation time and neuropathologyprofiles. In the Examples provided above it was shown that differentspecies and strains of prions displayed varying susceptibilities tobranched polyamine-induced disaggregation in vitro (FIG. 4A). Theseresults indicate that a polyamine-based in vitro protease digestionassay could be used as a simple and rapid diagnostic method for prionstrain typing. Currently, it is very difficult to remove prions fromskin, tissues, organs, blood, clothes, surgical instruments, foodstuffs,and surfaces. Standard prion decontamination requires either prolongedautoclaving or exposure to harsh protein denaturants such as 1N NaOH or6M guanidine thiocyanate. Branched dendrimers are non-toxic andrelatively inexpensive. These compounds are suitable for use as asterilizing agent to limit the commercial and iatrogenic spread of priondisease.

Example 15

[0333] Results provided here show that acidic conditions (<pH 5) enhancethe ability of other compounds (e.g., dendrimers, SDS, and urea) todenature PrP^(Sc) and destroy prion infectivity. Specific resultsdemonstrate that acidic conditions can be used to formulate effectiveprion disinfectants emphasizing the importance of acidic conditions.

[0334] The Effectiveness of 1% SDS at pH 3.4 to Eliminate PrP^(Sc) at 5Minutes or 2 Hours

[0335] A 2% homogenate of scrapie Sc237 brain in water was prepared byrepeated extrusion through a 22 G needle. Nuclei were removed bycentrifugation for 5 min at 1000 rpm. The clarified homogenate wasdiluted 2-fold and incubated for (FIG. 8A) 2 hrs or (FIG. 8B) 5 min at37° C. under the following conditions:

[0336] 1. 1% NP40, 50 mM sodium acetate pH 7.0;

[0337] 2. 1% NP40, 0.5% acetic acid pH 3.4;

[0338] 3. 1% SDS, 50 mM sodium acetate pH 7.0;

[0339] 4. 1% SDS, 0.5% acetic acid pH 3.4;

[0340] 5. 1% Sarkosyl, 50 mM sodium acetate pH 7.0; and

[0341] 6. 1% Sarkosyl, 0.5% acetic acid pH 3.4.

[0342] Plus (+) lanes indicate samples subjected to limited proteolysiswith 20 μg/ml proteinase K for 1 hr at 37° C., Minus (−) lanes indicatesamples not subjected to proteolysis. All samples were boiled in SDSsample buffer for 5 min prior to SDS polyacrylamide gel electrophoresis.Following transfer to Millipore Immobilon transfer membrane, developmentof the immunoblot was performed with d13 primary Fab.

[0343] The Effect of Temperature on the Ability of 1% SDS at pH 3.4 toEliminate PrP^(Sc)

[0344] A 2% homogenate of scrapie Sc237 brain in water was prepared byrepeated extrusion through a 22 G needle. Nuclei were removed bycentrifugation for 5 min at 1000 rpm. The clarified homogenate wasdiluted 2-fold and incubated for 5 min under the following conditions:

[0345] 1. 1% SDS, 0.5% acetic acid pH 3.4 at 4° C.;

[0346] 2. 1% SDS, 0.5% acetic acid pH 3.4 at 20° C.;

[0347] 3. 1% SDS, 0.5% acetic acid pH 3.4 at 37° C.; and

[0348] 4. 1% NP40, 0.5% acetic acid pH 3.4 at 20° C.

[0349] Plus (+) lanes of FIG. 9 indicate samples subjected to limitedproteolysis with 20 μg/ml proteinase K for 1 hr at 37° C. Minus (−)lanes indicate samples not subjected to proteolysis. All samples wereboiled in SDS samples buffer for 5 min prior to SDS polyacrylamide gelelectrophoresis. Following transfer to Millipore Immobilon transfermembrane, development of the immunoblot was performed with d13 primaryFab.

[0350] The Ability of Acidic Conditions to Enhance Urea-MediatedPrP^(Sc) Denaturation

[0351] A 2% homogenate of scrapie Sc237 brain in water was prepared byrepeated extrusion through a 22 G needle. Nuclei were removed bycentrifugation for 5 min at 1000 rpm. The clarified homogenate wasdiluted 2-fold and incubated for 2 hours with 0.5% NP40 plus urea and 50mM sodium acetate buffer (urea concentration and pH are indicated aboveFIG. 10).

[0352] All samples were subjected to limited proteolysis with 20 μg/mlproteinase K for 1 hr at 37° C., and boiled in SDS samples buffer for 5min prior to SDS polyacrylamide gel electrophoresis. Following transferto Millipore Immobolin transfer membrane, developments of the immunoblotwas performed with d13 primary Fab.

Example 16 SDS/Acetic Acid Formulation

[0353] Samples of 1% Syrian hamster brain homogenate containing 10⁷ LD₅₀units prion infectivity/ml were incubated with either 50 mM Tris acetatepH 7.0 or 0.5% acetic acid in the presence of either 1% NP-40 or 1% SDSfor 2 h at 37° C. Following incubation, each sample was inoculatedintracerebrally into 8 separate Syrian hamsters for a scrapie incubationtime assay. The results are shown below: Sample LD₅₀/ml 1% NP40, 50 mMTris acetate pH 7.0  10⁷ 1% NP40, 5% acetic acid, pH 3.6  10⁷ 1% SDS, 50mM Tris acetate pH 7.0  10⁵ 1% SDS, 5% acetic acid, pH 3.6 <10²

[0354] The above results clearly demonstrate that a formulationcomprised of approximately 1% SDS and approximately 0.5% acetic acid iseffective in inactivating prions. Such a formulation could provide foran extremely valuable commercial formulation due to the readyavailability of both acetic acid and SDS. However, those skilled in theart will recognize that other effective acids and effective detergentswith structure similar to SDS could be formulated to obtain the same orsimilar results. With respect to the acid component what is important isto create a formulation which keeps the pH of the formulation acidic andpreferably below 5.0 and more preferably below 4.0. The detergentcomposition need not be SDS. For example, the sodium component of thedetergent could be any cation such as calcium, lithium, potassium,magnesium etc. Further, the sulfate component could be substituted withchemically equivalent moieties. Sodium dodecyl sulfate includes ahydrocarbon component with eleven CH₂ groups terminated by a CH₃ group.Various other alkyl groups such as other straight-chained, branched orcyclic groups could be utilized. The alkyl moiety could contain from 2to 40 carbons and more preferably contains approximately 6-12 carbonatoms. The formulation can be added to appropriate solvents, inappropriate concentrations.

[0355] Further, the concentration of the formulation can be changedimmediately prior to use and thus sold at a highly concentratedformulation or sold in a concentration ready for use without dilutionwith a solvent such as water or alcohol. Still further, as indicatedabove the formulations of the invention can be supplemented withappropriate antibacterial and/or antiviral components as well ascomponents which inactivate other pathogens including parasites so thatthe final formulation is effective in killing or inactivating a widerange of infectious components.

Example 17 Effect of Detergent and pH on Protease-Resistant PrP^(Sc)

[0356] Samples of 1% Sc237-infected SHa brain homogenate were incubatedfor 15 min at 37° C. with detergent at a range pH values as indicated.Fifty millimolar sodium acetate buffers were used to maintain pH values3-6, and 50 mM Tris acetate buffers were used to maintain pH values7-10. The final pH value of each sample denoted above the correspondinglanes was measured directly with a calibrated pH electrode (Radiometer,Copenhagen). All samples were neutralized by addition of equal volume 4%Sarkosyl, 100 mM HEPES pH 7.5, 200 mM NaCl and subjected to limitedproteolysis with 20 μg/ml proteinase K for 1 h at 37° C. Apparentmolecular weights based on migration of protein standards are 30 and 27kDa. All samples were neutralized by addition of equal volume 4%Sarkosyl, 100 mM HEPES pH 7.5, 200 mM NaCl. Minus (−) symbol denotesundigested, control sample and plus (+) symbol designates samplesubjected to limited proteolysis with 20 μg/ml proteinase K for 1 h at37° C. Apparent molecular weights based on migration of proteinstandards are 30 and 27 kDa. FIG. 11 shows that SDS denatures PrPSc atpH<5 or pH>10.

Example 18 Characterization of PrPSc Denaturation Mediated by Acidic SDS

[0357] As shown in FIG. 12, samples of 1% Sc237-infected SHa brainhomogenate were incubated for 15 min at 37° C. in 1% SDS plus (lane 1)50 mM Tris acetate pH 7.0, (lane 2) 50 mM sodium acetate pH 3.6, (lane3) 50 mM glycine pH 3.7, and (lane 4) 0.2% peracetic acid, pH 3.4.Following incubation, an equal volume of 4% Sarkosyl, 100 mM HEPES pH7.5, 200 mM NaCl was added to neutralize each sample. Minus (−) symboldenotes undigested, control sample and plus (+) symbol designates samplesubjected to limited proteolysis with 20 μg/ml proteinase K for 1 h at37° C. Apparent molecular weights based on migration of proteinstandards are 30 and 27 kDa. The results in FIG. 12 show that SDSdenatures PrP^(Sc) under acidic conditions in different acidic buffers,including peracetic acid (a commonly used hospital disinfectant).

Example 19 PrP^(Sc) Denaturation by 1% Alkyl Sulfates and Sulfonates

[0358] As shown in FIG. 13A, different alkyl sulfates and alkylsulfonates were tested to see if they had the ability to denatureprions. 1% (w/v) Sc237 scrapie-infected Syrian hamster (SHa) brainhomogenate in 0.5% acetic acid, was incubated with 1% of the detergentspecified for 2 hours at 37° C. Following incubation, each sample wasmixed with equal volumes of 4% Sarkosyl, 100 mM HEPES (pH 8.0), and 200mM NaCl. The mixture was subjected to SDS-PAGE and immunoblot with d13chimeric Fab. Apparent molecular weights based on migration of proteinstandards are 30 and 27 kDa.

[0359] The results show that seven of the compounds tested wereeffective in denaturing prions. Those compounds are: C₁₀ alkyl sulfate,sodium salt, C₁₁ alkyl sulfate, sodium salt; C₁₂ alkyl sulfate, sodiumsalt (SDS); C₁₀ alkyl sulfonate, sodium salt; C₁₁ alkyl sulfonate,sodium salt; C₁₂ alkyl sulfonate, sodium salt; and C₁₃ alkyl sulfonate,sodium salt.

Example 20 PrP^(Sc) Denaturation by Alkyl Sulfates at 4° C. and 37° C.

[0360] As shown in FIG. 13B, different alkyl sulfates and alkylsulfonates were tested to see if they had the ability to denatureprions. 1% (w/v) Sc237 scrapie-infected Syrian hamster (SHa) brainhomogenate in 0.5% acetic acid, was incubated with 1% of the detergentspecified for 2 hours at 37° C. Following incubation, each sample wasmixed with equal volumes of 4% Sarkosyl, 100 mM HEPES (pH 8.0), and 200mM NaCl. The mixture was subjected to limited proteolysis with 20 μg/mlproteinase K for 1 hour at either 4° C. or 37° C., as indicated. Eachsample was subjected to SDS-PAGE and immunoblot with d13 chimeric Fab.Apparent molecular weights based on migration of protein standards are30 and 27 kDa.

[0361] Specifically, referring to FIG. 13A, lane 1 is C₅ alkyl sulfate,sodium salt, Lane 2 is C₆ alkyl sulfate, sodium salt, Lane 3 is C₇ alkylsulfate, sodium salt, Lane 4 is C₈ alkyl sulfate, sodium salt, Lane 5 isC₉ alkyl sulfate, sodium salt, Lane 6 is C₁₀ alkyl sulfate, sodium salt,Lane 7 is C₁₁ alkyl sulfate, sodium salt, Lane 8 is C₁₂ alkyl sulfate,sodium salt, Lane 9 is C₁₃ alkyl sulfate, sodium salt, Lane 10 is C₁₄alkyl sulfate, sodium salt, Lane 11 is an untreated control sample, Lane12 is an untreated control sample, Lane 13 is C₆ alkyl sulfonate, sodiumsalt, Lane 14 is C₇ alkyl sulfonate, sodium salt, Lane 15 is C₉ alkylsulfonate, sodium salt, Lane 16 is C₁₀ alkyl sulfonate, sodium salt,Lane 17 is C₁₁ alkyl sulfonate, sodium salt, Lane 18 is C₁₂ alkylsulfonate, sodium salt, and Lane 19 is C₁₃ alkyl sulfonate, sodium salt.

[0362] Specifically, referring to FIG. 13B, lane 1 is an untreatedcontrol sample at 4° C., Lane 2 is C₁₀ alkyl sulfate, sodium salt at 4°C., Lane 3 is C₁₁ alkyl sulfate, sodium salt at 4° C., Lane 4 is C₁₂alkyl sulfate, sodium salt at 4° C., Lane 5 is C₁₂ alkyl sulfate,lithium salt at 4° C., Lane 6 is C₁₂ alkyl sulfate, Tris salt at 4° C.,Lane 7 is an untreated control sample at 37° C., Lane 8 is C₁₀ alkylsulfate, sodium salt at 37° C., Lane 9 is C₁₁ alkyl sulfate, sodium saltat 37° C., Lane 10 is C₁₂ alkyl sulfate, sodium salt at 37° C., Lane 11is C₁₂ alkyl sulfate, lithium salt at 37° C., Lane 12 is C₁₂ alkylsulfate, Tris salt at 37° C., and Lane 13 is docusate, sodium salt at37° C.

[0363] The results show that ten of the compounds tested were effectivein denaturing prions. Those compounds are: C₁₀ alkyl sulfate, sodiumsalt at 4° C.; C₁₁ alkyl sulfate, sodium salt at 4° C.; C₁₂ alkylsulfate, sodium salt at 4° C.; C₁₂ alkyl sulfate, lithium salt at 4° C.;C₁₂ alkyl sulfate, Tris salt at 4° C.; C₁₀ alkyl sulfate, sodium salt at37° C.; C₁₁ alkyl sulfate, sodium salt at 37° C.; C₁₂ alkyl sulfate,sodium salt at 37° C.; C₁₂ alkyl sulfate, lithium salt at 37° C.; andC₁₂ alkyl sulfate, Tris salt at 37° C.

Example 21 Acidic SDS Disinfects Stainless Steel Wire Contaminated WithPrions

[0364] A system was devised to measure disinfection of cylindricalstainless steel suture wire (see Zobeley et al. (1999) Mol. Med.5:240-243). Zobeley et al. showed that prions bound to stainless steelwere resistant to disinfection by formaldehyde.

[0365] In the present example, the procedure of Zobeley et al. wasmodified to test the ability of acidic SDS to disinfect prions attachedto a steel surface. Five ml segments of 3-0 stainless steel suture wire(Ethicon) coated with RML (Rocky Mountain Laboratory) murine prions wereincubated in PBS or 10% SDS, 5% acetic acid at 65° C. for 16 h.Following incubation, the wire segments were implanted into the parietallobes of Tg(MoPrP)4053 indicator mice for the purpose of generating abioassay. The wires remained embedded in the brains of the indicatormice for the duration of the experiment.

[0366] Bioassays in Tg(MoPrP)4053 mice are able to detect as few as tenLD₅₀ infectious prion units/sample. The results, shown below in Table 3,indicate that incubation with acidic SDS reduces prion infectivity to alevel below detection of this sensitive bioassay. TABLE 3 Scrapieincubation time Prion infectivity Implanted wire (days ± SEM) n/n₀ (LD₅₀units/wire) Coated with RML prions and 74 ± 1 20/20 10³ incubated withPBS Coated with RML prions and >110  0/20 undetectable incubated withacidic SDS Control (no prions) >110  0/15 undetectable

Example 22 Acidic SDS Denatures PrP^(Sc) in BSE-infected Bovine Brain

[0367] It was investigated whether acidic SDS could denature prionstrains other than the Syrian hamster Sc237 strain. Samples wereincubated that contained 2.5% BSE-infected bovine brain homogenate withacidic SDS or control buffers for 15 min. at 37° C. In these conditions,PrP^(Sc) in BSE-infected brain tissue was denatured successfully byexposure to acidic SDS, as shown by Western blot in FIG. 14.

[0368] The buffers used in lanes 1-4 of FIG. 14 are as follows: lane1-1% NP40; 50 mM Tris acetate; lane 2-1% NP40; 0.5% acetic acid, pH 3.6;lane 3-1% SDS; 0.5% acetic acid, pH 3.6; and lane 4-1% SDS, 50 mM Trisacetate, pH 7.0. Following incubation, an equal volume of 2% NP40, 100mM HEPES, pH 7.5, and 200 mM NaCl was added to neutralize each sample,and then the samples were dialyzed (mol. wt. cutoff: 10 kDa) against 1%NP40, 50 mM HEPES, pH 7.5, 100 mM NaCl at 4° C. for 16 h. The minus (−)symbol above the lane indicates undigested, control sample. The plus (+)symbol above the lane indicates the sample was subjected to limitedproteolysis with 20 μg/ml proteinase K for 1 h. at 37° C.Protease-digested samples were centrifuged at 100,000×g., supernatantfractions were discarded, and pellets were boiled for 10 min. in SDSsample buffer prior to electrophoresis. Apparent molecular weights basedon migration of protein standards are 30 and 17 kDa.

Example 23 Basic SDS Renderss Prions Non-Infectious

[0369] Bioassay data confirm than an aqueous formulation of 1% sodiumdodecyl sulfate (SDS) at pH 10 inactivates prion infectivitiy, asoriginally suggested by Western blot assays detecting PrP^(Sc).Incubation Time Inocula (days ± S.E.M.) n/n₀ pH 10 control, 5 min, 25°C. 81 ± 1 12/12 pH 10 control, 2 hrs, 25° C. 79 ± 1 12/12 pH 10 control,5 min, 37° C. 78 ± 1 12/12 pH 10 control, 2 hrs, 37° C. 82 ± 2 12/12 1%SDS, pH 10, 5 min, 25° C. 101 ± 1  12/12 1% SDS, pH 10, 2 hrs, 25°C. >141  4/12 1% SDS, pH 10, 5 min, 37° C. 104 ± 2  12/12 1% SDS, pH 10,2 hrs, 37° C. >164  3/12

[0370] All inocula were 1% Sc237 brain homogenate samples in 50 mM Trisacetate pH 10.0 plus or minus 1% SDS shaken in vitro under the indicatedconditions. 0.1 ml was added to 0.9 ml diluent for bioassay of scrapieinfectivity and inoculated into Syrian hamsters intracerebrally.

Example 24 Acid SDS at 65° C.

[0371] Acidic SDS denatures Sc237 prion infectivity rapidly andcompletely at 65° C. After 229 days, no hamsters (0/12) inoculated with1% Sc237 brain homogenate treated in vitro with 1% SDS, 0.5% acetic acidfor 15 minutes at 65° C. have developed neurological disease. Twoanimals were removed from the experiment after fighting each other, andthe ten remaining animals are alive and healthy. Thus, lukewarmtemperature optimizes the prion disinfecting ability of acidic SDS, evenwith a relatively short exposure period.

[0372] While the present invention has been described with reference tothe specific embodiments thereof, it should be understood by thoseskilled in the art that various changes may be made and equivalents maybe substituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

That which is claimed is:
 1. A method of rendering infectious proteinsnon-infectious, comprising: contacting infectious proteins with aformulation comprising a salt of an alkyl sulfate; allowing the proteinsto remain in contact with the salt of the alkyl sulfate at a temperaturein a range of from about 10° C. to about 80° C. for period of time andunder conditions so as to render the proteins non-infectious.
 2. Themethod of claim 1, wherein the infectious proteins are infectious prionproteins.
 3. The method of claim 1, wherein the formulation is anaqueous formulation comprising 0.25% or more of the salt of alkylsulfate.
 4. The method of claim 1, wherein the salt of alkyl sulfate isa salt of a cation of a metal selected from the group consisting ofsodium, calcium and magnesium.
 5. The method of claim 1, wherein thesalt of alkyl sulfate is sodium dodecyl sulfate.
 6. The method of claim1, wherein the period of time to render the protein non-infectious istwo hours or less.
 7. The method of claim 6, wherein the period of timeto render the proteins non-infectious is one hour or less.
 8. The methodof claim 1, wherein the conditions comprise a pH selected from the groupof ranges consisting of (a) less than 5.0 and (b) more than 9.0.
 9. Themethod of claim 1, wherein the conditions comprise a pH of 4.0 or less.10. The method of claim 1, wherein the conditions comprises a pH of 10.0or more.
 11. The method of claim 1, wherein the alkyl moiety iscomprised of from 2 to 40 carbon atoms.
 12. The method of claim 11,wherein the alkyl moiety is comprised of from 6 to 12 carbon atoms. 13.The method of claim 1, wherein the conditions comprise a temperature ina range of from about 15° C. to about 70° C.
 14. The method of claim 1,wherein the conditions comprise a temperature of about 30° C.±15° C. 15.The method of claim 1, wherein the formulation comprises 1% or more ofthe salt of the alkyl sulfate.
 16. The method of claim 1, wherein theformulation comprises 3% or more of the salt of the alkyl sulfate.
 17. Amethod of rendering infectious proteins non-infectious, comprising:contacting infectious proteins with a formulation comprising a salt ofan alkyl sulfate; allowing the proteins to remain in contact with thesalt of the alkyl sulfate at a pH of 5.0 or less for period of time andunder conditions so as to render the proteins non-infectious.
 18. Themethod of claim 17, wherein the infectious proteins are infectious prionproteins wherein the formulation is an aqueous formulation comprising0.25% or more of the salt of alkyl sulfate and further wherein the saltof alkyl sulfate is a salt of a cation of a metal selected from thegroup consisting of sodium, calcium and magnesium.
 19. The method ofclaim 17, wherein the salt of alkyl sulfate is sodium dodecyl sulfate.20. The method of claim 17, wherein the period of time to render theprotein non-infectious is two hours or less and the pH is 4.0 or less.21. The method of claim 20, wherein the period of time to render theproteins non-infectious is one hour or less.
 22. The method of claim 17,wherein the conditions comprise a temperature in a range of from about15° C. to about 140° C.
 23. The method of claim 24, wherein theconditions comprise a temperature of about 132° C.±10° C.
 24. A methodof rendering infectious proteins non-infectious, comprising: contactinginfectious proteins with a formulation comprising a salt of an alkylsulfate; allowing the proteins to remain in contact with the salt of thealkyl sulfate at a pH of 9.0 or higher for period of time and underconditions so as to render the proteins non-infectious.
 25. The methodof claim 24, wherein the infectious proteins are infectious prionproteins and wherein the formulation is an aqueous formulationcomprising 0.25% or more of the salt of alkyl sulfate and furtherwherein the salt of alkyl sulfate is a salt of a cation of a metalselected from the group consisting of sodium, calcium and magnesium. 26.The method of claim 24, wherein the salt of alkyl sulfate is sodiumdodecyl sulfate.
 27. The method of claim 24, wherein the period of timeto render the protein non-infectious is two hours or less and the pH is10.0 or higher.
 28. The method of claim 27, wherein the period of timeto render the proteins non-infectious is one hour or less.
 29. Themethod of claim 24, wherein the conditions comprise a temperature in arange of from about 15° C. to about 140° C.
 30. The method of claim 24,wherein the conditions comprise a temperature of about 132° C.±10° C.31. A formulation, comprising: a salt of an alkyl sulfate present in anamount in a range of from about 0.25% to 20% by weight; an acid presentin a molarity sufficient to maintain the formulation's pH at about 4.5or less; and a solvent.
 32. The formulation of claim 30, wherein thesalt of the alkyl sulfate is sodium dodecyl sulfate.
 33. The formulationof claim 30, wherein the acid selected from the group consisting ofperacetic acid and is acetic acid.
 34. The formulation of claim 30,wherein the solvent is selected from the group consisting of water,ethanol and methanol.
 35. A formulation, comprising: a salt of an alkylsulfate present in an amount in a range of from about 0.25% to 20% byweight; a base present in a molarity sufficient to maintain theformulation's pH at about 9.5 or more; and a solvent.
 36. Theformulation of claim 35, wherein the salt of the alkyl sulfate is sodiumdodecyl sulfate.
 37. The formulation of claim 35 wherein the base issodium hydroxide.
 38. The formulation of claim 35, wherein the solventis selected from the group consisting of water, ethanol and methanol.